MICRO LAB

Slant or Plate Culture

1. Place a loopful of water in the center of the slide. With the inoculating needle, aseptically pick up a very small amount of culture and mix into the drop of water. 2. Spread this out to about ½ inch area. 3. Allow the slide to air dry, or place it about 12 inches above the flame to dry in hot air.

Once your microscope is in Kohler illumination, you should not adjust the field diaphragm, Condenser knob, or Condenser Centering knobs.

True

Bacterial Identification; Aerobic Gram Positive Cocci

Pure Culture (Isolation) Colony Characteristics Colony Morphology Hemolysis No hemolysis Complete hemolysis Incomplete hemolysis Double Zone hemolysis Gram Stain Gram reaction Cellular Morphology and arrangement Biochemical Properties Antigenic Properties

Anaerobic Carbohydrate Catabolism

dont mem

Aerobic Carbohydrate Catabolism

dont mem CAC-> ETC-> O2-> H2O

Bacterial metabolism is the sum of:

- Anabolic processes (synthesis of cellular constituents which requires energy). - Catabolic processes (breakdown of cellular constituents with concomitant release of waste products and energy-rich compounds).

Bacterial Growth (an example using a rod-shaped cell)

1. Cell elongates and DNA is replicated. 2. Cell wall and plasma membrane begin to divide. 3. A crosswall forms betweenand completely separates the replicated DNA masses. 4. Cells separate (most organisms).

Double Zone Hemolysis

A very limited number of organisms will produce Double Zone Hemolysis. • Effectively, 2 different enzymes diffuse out from the colonies into the medium at different rates . • The first enzyme diffuses through the agar rapidly and lyses the rbc's . • The second enzyme diffuses more slowly and breaks down the hemoglobin. Staphylococcus aureus, Staphylococcus pseudintermedius, and Clostridium perfringens are capable of producing Double Zone Hemolysis. Note: Clostridium perfringens is an Obligate Anaerobe. It can only grow under anaerobic conditions and will, therefore, produce double zone hemolysis only under anaerobic conditions. limited number of organisms*** So we call it a zone of complete hemolysis around by incomplete sometimes it's called a zone of beta hemolysis is surrounded by alpha prime, not quite the same thing is incomplete hemolysis when we're just talking about simple incomplete hemolysis in this case the hemoglobin is not going to turn to methemoglobin the first set of enzymes goes out breaks up in the blood cells. and the second thing that comes along and starts breaking down the hemoglobin before it gets a chance to break down on its own to methemoglobin. when we're just talking about simple incomplete analysis in this case the hemoglobin is not going to turn to methemoglobin In some cases the outer zone will be lighter than the surrounding medium in some cases, a little bit darker depending on the strain and the actual. and Exact conditions, it was growing under but there's two distinct zones around the colonies, which i'm trying to indicate on my pictures with my arrows is actually pictures Staphylococcus aureus, Staphylococcus pseudintermedius, and Clostridium perfringensare capable of producing Double Zone Hemolysis .Note: Clostridium perfringensis an Obligate Anaerobe. It can only grow under anaerobic conditions and will, therefore, produce double zone hemolysis only under anaerobic conditions. duck v human ex E coli - primary streak is dark and are defecating in media and discolor media ex: step pyr- colony centered over each circle and that is producing meolyis and break open RBCs and break up Hb to create a tearing of the media/clearing of zone; zone same diameter of the edge of hemolysis is the same distance out from the edge of each colony where there's lots of colonies lined up together. **the zones and analysis run together but they're the same size. streptococcus pneumoniae- dark green haze at isolated colonies -we were to scrape those off you'd actually see a green dot remaining in the medium -where the bacteria have broken under the blood cells released the hemoglobin and the hemoglobin By sitting in the air and the light has broken down just broken down the methemoglobin by itself. *still almost the same just a minor chemical change that happens spontaneously in presence of oxygen and light the methemoglobin turns green and a hemolysis double hemolysis- staph aureus; some around each awith complete hemolysis and around another zome **staph pseudmeinais and clostridium perfringens are three examples of organisms that can produce double hemolysis **pure culture before identification; if not then subculture 1. broth- e coli and staph aureus; goal of incoluating broth from broth ; get in suspension shake *medium is clear and other is turbid because some is motile and others are not ; settled at bottom= turbitdity 2. small slant tube- growth on surface of tube; used needle and wiggled to top on surface, mixed. culture cant tell two colony types; not used to isolated but used to get some growth or find chemical reaction 3. solid media- same effect 4. deep slant; stab bottom of tube and along stab there is turbidity to show growth Okay, we wouldn't use this to try to determine we had mixed culture, if you did see multiple content types on something like this you'd say I better go back and strict for isolation to get a pure culture, then I can go back to my individual media and identify the organism.So getting into the blood of the tube and on the slant was the purpose of using a needle stabbing to the but streaking the slant. we had motility medium, these are your plates and auger slants are.Have about 15% auger added to them so they're a nice firm surface for the bacteria to grow on or a firm meeting for the bacteria to grow in we stab. *motility medium has about half a percent or 1% auger making it semi solid it's not completely impervious to the bacteria swimming through the medium. cloudiness tribute it in our motility medium is evidence of growth in that part of the medium. nonmotile organism grows only along the stab and doesn't swim out to the size of the tube if you were to stab and not go straight, you might see a bit of a you could see where you stabbed it with a little wider it gets a little squirrely. *But there's nice clear medium around the growth, you could potentially read letters around the growth see past it and see letters. With a mtile organism that organism has flagella and can swim and swims out toward the walls of the tube so the entire tube in this case is turbid or cloudy. *Often, with this kind of organism, this is E. coli, as our example often when you have an organism that's mobile mobile mobile swims grows out, besides the two rgoups throughout the entire medium you'll often get darker read or more read at the top of the two, and then a pale area target, but not quite read. It basic pale pink and the the bottom is more pink than that little layer and surface I don't know why they do that, they just do. it just differences environmental conditions in different parts of the tube so they grow a little bit differently, but we have pink all the way out to the side, so that organism is motal **we're not going to use motility medium to determine the organisms oxygen new apartments, but you can potentially see some variations on these themes. *stab there's a couple of bubbles down in there were when I stabbed the tube took some oxygen and made a couple of bubbles. If it were motile and required oxygen couldn't grow without oxygen you may actually see a red layer across the surface of the medium.Because it swims across the surface, but where there's no oxygen can grow, so you might see the stab is read partway all the way down the tube. If there's read out to the sides of the tube or pink out to the sides of to tube it's motile if it's only growing along the stab it's not motile

Kirby-Bauer Antimicrobial Susceptibility Testing

Empirical Treatment is common and often all that is needed (in English, "This usually works in cases like this. Let's give it a try. If it doesn't work, we'll do some tests." In many, perhaps most, cases, when a clinician determines that a patient needs treatment with an antimicrobial drug, he/she prescribes a drug with a broad spectrum of activity that is likely to be effective against whatever organisms usually cause the kind of infection the patient has. If the patient gets better, the clinician made the right choice. If the patient does not improve, a sample may be sent to the Diagnostic Lab for culture and sensitivity testing. Quick Note In the past, almost any time a child, sometimes an adult, was brought to the Pediatrician/Doctor, the Doctor would write a prescription for an antibiotic, "Just in case". We prefer to avoid unnecessary use of antibiotics now. Overuse of antibiotics contributes to the emergence of drug resistant, even multi-drug resistant strains of bacteria. Viruses are not affected by antibiotics, so if a patient is suspected of having a viral infection, why waste perfectly good drugs. Empirical Treatment didn't cut it. Now what? If we are going to start running tests, we will usually want to identify the infectious organism and decide what drug is the best choice for treatment. Rapid identification systems is a separate presentation. • Remel RapID One is the example we use in this class. The Kirby-Bauer Antimicrobial Susceptibility Test provides clinicians with information to assist in selection of an appropriate antimicrobial drug. ֎Minimum Inhibitory Concentration is a more commonly used test now, but it is more complicated to run and more difficult to explain, so we use the simpler system to make the point. ֍Terminology reminder: An antimicrobial compound (drug) is a chemical that inhibits the growth of, or kills, one or more microorganisms. It may be synthetic or naturally produced. An antibiotic is an antimicrobial compound produced by a living organism. The basic concept of the test: A lawn of bacteria is created on the surface of an agar plate. Paper discs impregnated with various antimicrobial compounds are dispensed onto the agar and the plate is incubated allowing the organism to grow, or not grow, in the presence of the drugs. The zones of inhibition of growth of the organism around the discs are measured and the measurements are compared to a chart. The report sent to the clinician lists the drugs tested and the susceptibility, or resistance, of the organism to each drug. We want to standardize our technique and inoculation so that we get repeatable results and can use standardized charts for interpretation. A standardized concentration of bacteria is used. Mueller-Hinton Agar is used. Some labs use Mueller-Hinton Blood Agar when working with fastidious microorganisms such as Streptococcus spp. Paper discs impregnated with known quantities of the chosen antimicrobial drugs are distributed evenly on the plate. The antimicrobial drugs diffuse into the medium as the organisms grow. Some people did a lot of work to determine the concentrations of each drug at various distances from the discs and correlate those concentrations to tissue concentrations in patients. The plates are incubated at 37C for 24 hours Even the thickness of the Mueller-Hinton Agar is important. Each drug will diffuse into a certain volume of medium. If the agar is too thin, the zones of inhibition will be larger because the drugs diffuse further horizontally. If the agar is too thick, the zones of inhibition will be smaller because the drugs diffuse further vertically.

Antigen Capture ELISA

If we wanted to detect Antigens in a sample, we might coat the wells with Antibodies specific for the Antigen we wanted to detect. If the Antigens of interest are present in the sample, they will bind. The 2o Antibodies used would be specific for the Antigen. Otherwise the process is similar

ELISA color change

Keep in mind: the color is dependent on the enzyme/substrate combination used. There are several enzyme/substrate combinations available

Köhler Illumination

Köhler illumination describes a method to ensure the light microscope used is setup in such a way so as to provide the best sample illumination. This method is named after August Köhler, the man who invented it. If the light path is set up properly, you will have the advantages of an evenly illuminated field, a bright image without glare and minimum heating of the specimen. The following instructions apply to any microscope, upright or inverted which is equipped for transmitted light bright field illumination. Focusing of the field diaphragm as discussed here should be done for phase and differential interference optics as well. 1. Switch on the light source and make sure that light is coming through the field diaphragm at the base (upright microscope) or the top (inverted microscope) of the microscope stand. It may help to place a piece of paper over the field stop to see the light. 2. Place your specimen on the stage and turn the nosepiece (which holds the objective lenses) to the 10X or 20X lens. Open the field diaphragm as far as it will go. 3. Notice whether or not your specimen is illuminated. It will help to place a piece of paper over the top of the specimen to see if light is getting through to it. If you are using the bright field condenser stop, open the iris diaphragm (or aperture diaphragm) on the condenser turret (which contains the stops for bright field and phase, etc.) wide open to give the maximum illumination. If there is a swing-in front lens for the condenser (directly above (inverted) or below (upright) the specimen), you may need to swing it into the light path. 4. Now bring your specimen into focus with the coarse and fine Focusing knobs. The best way to do this is to rack the lens as close possible to the specimen watching the objective lens all the time (and NOT looking into the oculars) to make sure that the lens does not run into the slide. Then rack the lens away from the stage (or vice versa) while looking through the oculars to bring the specimen into focus (details are as sharp as they can be). If the light is too bright, reduce it with the rheostat on the light source. 5. When the specimen is in focus, start to close the field diaphragm and also begin to carefully move the condenser up and down with the condenser Focusing knobs. Look for a sharp image of the edge of the field diaphragm. This may be a little with a long working distance condenser. Also, if the iris diaphragm in the condenser turret is open wide, the glare may obscure the edge of the field diaphragm silhouette somewhat. Furthermore, you may find that this edge is not centered. 9 6. When the edge of the field diaphragm silhouette is sharply defined, center it with the two knobs (usually knurled knobs) coming out diagonally from the condenser. Close down the field diaphragm most or all the way to get it centered properly. When it is centered, open the field diaphragm until its edge is outside the field (Figure 3). If you are doing brightfield or differential interference microscopy, do not yet open the field diaphragm. 7. As stated before, you may notice some glare around the edge of the field diaphragm, that the edge area outside the edge is not completely dark like the outer part of the whole field as you should see it now. This glare comes from light bouncing around in the light path and going in and illuminating the specimen in such a way as to obscure detail in the specimen. To reduce this glare, close down the iris aperture in the condenser turret until all of the dark area outside of the field stop silhouette is evenly dark. Now open up the field diaphragm until the edge of the diaphragm silhouette is outside the field of view. You should also now be able to turn up the light at the power source. 8. Your specimen should be properly illuminated and should give you a great image. If it does not, check to make sure your lenses and other optical components are clean. Then recheck to see that you have followed each step properly

Enriched media Selective media Differential media

Media fortified with blood, yeast extracts, or brain or heart infusions are useful in growing fastidious organisms. Media that inhibit growth of certain microorganisms, while allowing the growth of selected organisms. Media that allow differentiation of microorganisms based on the latter's biochemical characteristics

Smear Preparation

Our slides have one frosted end and 2 circles etched onto them. • Label your slide (what is going on the slide) . • If you are making a smear from broth medium, ensure that the bacteria are in suspension, then just transfer a drop (or 2) of the broth to the slide • If you are making a smear from a colony on solid medium, you need to add a small drop of water to the slide first. • Spread your sample out to at least the diameter of the circle. • A thinner smear is a better smear. The bacteria are more likely to stain correctly. You are more likely to be able to see individual cells For most staining techniques, you need to air dry your slide, then heat fix the bacteria to the slide. • The heat blocks are set to around 100 F (40C). - You want the slide to dry, not cook. • After your slide is dry, set it on the shelf above the bacti-cinerator for 40 - 45 seconds. - You want to heat the slide enough that the surface proteins on the bacteria will stick to the glass. If you overheat your slide during the air drying or heat fixing steps, you risk distorting the shape of the microorganisms or rupturing the cells. If you don't heat fix, your specimen is liable to wash off the slide during staining and, well, then you have to start over instruments- loop, inoculating needle put in tube rack ( store with ends up in air), sterilize in bactoincenerator BROTH 1. grab slide 2. label with pencil and not pen/sharpie because stains are alcohol based and will remove pen/sharpie 3. keep frosted surface of slide as top, has 2 etched circles and end, put smear in circles ( makes easier to find with microscope) 4. both medium will be thin and not visible until under microscoope 5. if culture settles, agitate it until its back in suspension ( dont shake up and down because caps are not air tight and cause a spill) 6. take incolulator loop put in bacuincenerator for 10-15 s keep hold of handle and pull out ( glowing) 7. pull cap off of tube, hold cap in pinky finger of hand with loop, sterilize top tube in front of incenerator, pull out loop of both, wave tube in front of incenerator, cap tube and set down 8. grab slide, put drop of both in circle and spread to size of circle, thin smear and dry quickly 9. resterilize loop ( all parts that go in tube; be careful of hot handle), store on bench tip facing up in tube rack or hole next to incinerator** never let go of handle SOLID MEDIUM 1. Bacillus serius 2. put drop of water on slide ( deionized water), barely touching slide with dropper, store back in rack 3. Sterilize inoculating loop, touch plate where no bacteria, sizzle, then touch colony 4. transfer to drop. of water on slide and spread to size of circle so it will dry faster 5. Sterilize loop, store **keep plate upside down to prevent rain on your parade BOTH-set slide on warmer (100 F), dry slide barely above room temp, overheat slide before dry specimen will become ruptured ( few minutes) 2. makes bacteria stuck on surface and melt surface proteins a little to stick but not too much to rupture 3. put on heat fixing on top of incinerator for 30s-40s-1 min 4. want to heat fix so bacteria dont slide off when staining 5. place slide on stain tray; stain slide with stain that requires heat fix stain

Bacterial Identification

Pure Culture -> Colony & Gram Stain Morphology-> Growth Characteristics, Biochemical Properties, and Antigenic Properties or subculture ones that look diff-> pure culture

Triple Sugar Iron (TSI) Agar

Purpose 1. To determine the ability of an organism to attack a specific carbohydrate(s) (gluc, lact, sucr) incorporated in a basal growth medium, with or without the production of gas, along with the determination of possible hydrogen sulfide (H2S) production . 2. Aids in identifying the atmospheric requirement of the bacteria which grow on/in the medium. 3. We will use TSIA in identification of a wide variety of organisms. It is especially useful for identification of members of the family Enterobacteriaceae. Procedure 1. With a straight inoculating needle, touch the top of a well-isolated colony. -sterilize, touch not hot, touch on colony 2. Inoculate the TSIA tube by stabbing through the center of the medium to the bottom of the tube and then streaking the surface of the agar slant as you withdraw the needle. -pull cap off, sterilize tube, stab to bottom and streak on way up, sterilize tube 3. Recap the tube, and incubate the tube at 35-37 ºC in a non-CO2 incubator for 18-24 hours. -sterilize loop and the handle 4. The slant and the butt of the tube are examined, and these results are recorded as fractions 'Slant/Butt'. Substrates: 0.1% Glucose 1% Lactose 1% Sucrose Sodium Thiosulfate Indicators: Phenol Red - pH Indicator Ferrous Sulfate - H2S Indicator • Red/Yellow [alkaline/acid] - The bacterium is a Facultative anaerobe, saccharolytic and utilizes glucose only. - The confinement of the yellow color to the butt of the tube is the result of the differential quantities of the sugars. - Abbreviation: K/A. Glucose +ve Facultative Anaerobe-> Peptone use on slant (aerobic), still using glucose in butt (anaerobic). • Yellow/Yellow [acid/acid] - The bacterium is facultative anaerobe, saccharolytic and utilizes, glucose and lactose and/or sucrose. - Abbreviation: A/A • Bubbles or cracks in the medium. - Indicate production of CO2 Gas. - The organism is Aerogenic. - Can only occur if the organism is saccharolytic. - Abbreviation: A/A+G Glucose +ve, Facultative Anaerobe-> Also uses Lactose &/or Sucrose Red/No Change [alkaline/alkaline] - Some organisms fail to ferment even glucose, and because they are strictly aerobic, they fail to grow *********** of the tube. - In these cases, the butt will be unchanged in color, and the slant either alkaline or unchanged. - Abbreviation: K/N Formation of a Black precipitate: - Hydrogen Sulfide is produced under Acidic, Anaerobic conditions - If an organism can produce H2S gas, the gas will diffuse out of the cells and react with the iron to form iron sulfide, which appears as a black precipitate. - It can mask any acid/alkaline results. Sulfur reduction requires an acidic environment, so if the black precipitate is present, some fermentation took place. That means the butt is Acidic, the organism is a saccharolytic Facultative Anaerobe. Formation of a Black precipitate (Hydrogen Sulfide production): - If the bottom of the slant is obscured by the precipitate, you should look at your Phenol Red Carbohydrate Broths (Glucose, Lactose, & Sucrose) to make a final determination as to which of the CHO 's the organism uses. - Any black precipitate indicates H 2S production. - The H 2S may, or may not obscure enough of the tube to make determination of Lactose/Sucrose use difficult. - Darkening of the medium at the surface of the slant is NOT H2S production. - Pseudomonas aeruginosa and some other bacteria produce pigment on an alkaline slant (no lactose or sucrose use) lactose broth 1. sterilize loop, pick up colony 2. sterilize top, incoluate, wiggle, sterilize loop and tube 4. cap *bacteria on wire so must sterilize sucrose *same procedure as glucose and lactose lactose- durham tube sucrose- no durham tube various colony morphology, many look similar tho -protease highly motile, touch just center of plate and dont streak for isolation procedure 1. label different colony for each TSI tube ( 7) 2. needle sterilize, touch colony on agar, uncap with pinky, sterilize, stab to bottom and streak on surface of slant 4. sterilize, cap, resterilize needle and handle; out on with a quart of of turn open so air in and and gas out 5. repeat for all

Anaerobic Carbohydrate Catabolism

The take home point: There are multiple pathways that different organisms may use for glucose fermentation and we can test for some of them. Mixed Acid Pathway -Mixed short chain fatty acids <4.4 -Various organic compounds -Methyl Red test -Methyl Red 2,3 Butanediol Pathway -2,3 Butanediol pH >4.4 -Acetoin (acetylmethylcarbinol) -Voges-Proskauer Reagents -VP-I: α-naphthol and KOH; VP-II: Creatine

Acid-Fast Stains

Ziehl Neelsen Stain Ziehl-Neelsen (the classic acid-fast stain) is used to identify organisms that have waxy material (mycolic acids) in their cell walls. The most clinically important acid-fast bacterium is Mycobacterium tuberculosis. Acid-fast staining 3 is reserved for clinical samples from patients suspected of having mycobacterial infection • A special bacteriological stain used to identify Acid-Fast organisms, mainly Mycobacterium spp. Acid fast organisms like Mycobacterium spp. contain large amounts of lipid substances within their cell walls called mycolic acids. These acids make the organisms resistant to staining by ordinary staining methods such as a Gram stain. ** have gram positive cell wall structure + mycolic acid; wild type org that is grown under ideal conditions with this layer and wont gram stain because cant get past mycolic aicid layer • The reagents used in Ziehl-Neelsen staining: - carbol fuchsin, - acid alcohol, and - methylene blue.; Acid-fast bacilli will be bright red after staining. • Initially, Carbol Fuchsin stains every cell. Heating the slide while Carbol Fuchsin is on the slide helps the primary stain penetrate the Mycolic acid layer in the cell wall of Acid-Fast organisms. • The slide is given a minute or 2 to cool before decolorizing. - This gives the Mycolic Acid layer a chance to re-solidify. - Also, if you put room temperature acid-alcohol on a hot slide, the slide may crack. • When the slide is decolorized with acid-alcohol, only non-acid-fast bacteria get decolorized since they do not have a thick, waxy lipid layer like acid-fast bacteria. • The mycolic acid in an acid-fast cell wall prevents the decolorizer from penetrating the cell and washing out the primary stain. • When the counterstain (Methylene Blue) is applied, non-acid-fast bacteria pick it up and become blue when viewed under the microscope. • The mycolic acid layer prevents the counterstain from entering the Acid- fast cells. Because the Acid-fast bacteria retain Carbol Fuchsin they appear red. * acid- fast= red * non acid- fast= blue Kinyoun Stain • An acid-fast staining procedure used to stain species of the genera Mycobacterium, Nocardia and Cryptosporidium species. • It involves the application of a primary stain (carbol fuchsin), a decolorizer (acid-alcohol), and a counterstain (methylene blue). • Unlike the Ziehl-Neelsen stain (Z-N stain), the Kinyoun method of staining does not require heating. - In the Ziehl-Neelsen staining technique, heat softens the mycolic acid layer, - In the Kinyoun staining technique, detergent (Tergitol) softens the mycolic acid layer to allow penetration of the primary stain. procedure Kinyoun- 1. Flood the entire slide with Kinyoun Carbol Fuchsin Stain .2. Allow the smear to stain for 2 minutes. 3. Rinse the slide with water. 4. Flood the slide with Acid-alcohol Decolorizer and decolorize until no more color drains from the slide (approximately 3 to 5 seconds). ** stronger than acetone alcohol and timing isnt as crucial a gram stain 5. Rinse the slide thoroughly with water and shake off any excess moisture. 6. Flood the slide with Counterstain (Methylene Blue or Brilliant Green) and allow the slide to stain for 30 seconds. ** will stain anything thats not acid fast 7. Rinse thoroughly with water and allow to air dry or blot dry with bibulous paper. 8. Examine the smear microscopically under a 100x objective. ; blue background and cells non-acid fast, red/pink for acid fast cells Procedure Ziehl-Neelson- 1. Prepare heat-fixed smears of the lymph nodes provided with acid and no-acid fast specimens. ( soldi media or broth culture use same procedure) 2. Place the slide on the staining rack. 3. Cover the smear with filter paper. Saturate the filter paper with carbolfuchsin. ** flood slide with carbolfushin and let sit for 2 minutes -detergent within carbolfushiin that can penetrate mycolic layer 4. Heat over a Bunsen burner till steam appears. Once steam appears, set aside till the steam disappears. Repeat for a total time of about 5 minutes. Do not allow the slide to dry out and avoid excess flooding! 4. Remove the slide, let it cool, and rinse with water for 30 seconds. 5. Decolorize with acid-alcohol mixture drop by drop until all excess stain is washed away. 6. Rinse with water for 5 seconds 7. Counterstain with methylene blue for1 minute. 9. Rinse with water for 5 seconds. 10. Blot dry with bibulous paper and examine under oil immersion. Acid-Fast bacilli will appear as red aggregations against a blue background (Figure 12)

Post lab Lab 7

aerotolerance testing - TGH binds O2 - location in tubes= O2 requirements e coli- faculative anaerobe streptocard- put colonies. in. extraction buffer and put in hot block RapID strip - peel off cover -add 3 drops 1 reagents in PRO, GGT, PYR - report strip sheet and. color guide - indole reagent 2 drops in well after reading ADON - compare - add up numbers - put in ERIC susceptibility - zone diameter of 15 mm - zone vary on type of organism -widness isnt an accurate representation; compare on chart - closest colony to center of disj and x2 for diameter - go through chart with measurements label antiserums-> lancefield identification - agitate suspension with latex beads -pipet onto each antisera - mix with toothpick - ones swirling in liquid is positive

Defined medium

is a medium for which we know exactly what it contains - every salt, protein, carbohydrate, lipid, or whatever else you can think of. Example: Simmon's Citrate Agar For most media, we don't have that level of knowledge of the exact composition.

Brightfield Microscopy Phase-contrast Microscopy

The specimen is stained, so its image appears dark against a brighter background. The light is usually provided through a tungsten filament lamp. In Phase-contrast microscopy, light beams are deflected by different thicknesses of the object. The light beams are reflected a second time when they strike the objective. The light waves increase in length when the specimen is of uniform thickness. By contrast, the light waves decrease when the specimen has different thicknesses and thus different refractive indices Phase-contrast microscopy is particularly useful for direct observation of specimens that are not stained, e.g., protozoa.

Pre lab notes

want a lawn of bacteria all on plate for a susceptibility test - impregnate with antimicrobial compounds os if drug inhibits growth of organism you can see that 1. label plate 2. grab bacteria with swab 3. streak entire plate with swab and cover entire surface 4. third of a turn and repeat streaking, repeat in a third direction 5. dispose swab in biohazard bag 6. squeeze spring to drop disk onto plate, repeat with other dispenser or use sterile forceps to pull disk out and put on plate 7. move disks in correct position with forceps 8.tap the disks to make sure they're stuck on the plate 9. leave in incubator and see if capable of growth in the presence of these drugs Bacetracin- strep biogenese optogin- step pneumonia cAMP test- 1. loop or toothpick pick up colony, label plate 2. touch plate and streak across the center of the plate 3. dont mix cultures and repeat toothpick procedure with other samples; touching near other sample at center and drag towards outside ( T) 4. repeat with other sample; can do a couple streaks 5. incubate 24-48 hours ( enhancement of hemolysis (strep galactiae) or no)

Remember from previous discussions

• Saccharolytic organisms can use glucose as a food source. For some saccharolytic organisms, glucose is the only carbohydrate they can use as a food source. Many saccharolytic organisms have the ability to use other carbohydrates, in addition to glucose, as food sources. • An easy way to think about it is, saccharolytic organisms can use any carbohydrate that they can enzymatically convert to glucose. • If a saccharolytic organism lacks an enzyme to convert a CHO to glucose, it can't use that CHO. Carbohydrates may be broken down by respiration or fermentation depending on the enzymes produced by the organism. In addition to glucose, Saccharolytic organisms can use peptones (partially digested proteins) as a food source • Asaccharolytic organisms cannot use glucose (or any other carbohydrates) as a food source. These organism use peptones as a food source. Peptone use only occurs under aerobic conditions. • So, all asaccharolytic microorganisms are Obligate Aerobes. • Saccharolytic microorganisms may be Obligate Aerobes, Facultative Anaerobes, Obligate Anaerobes, Aerotolerant Anaerobes, or Microaerophiles.

microscope

use handles to move microscope out of cabinet 1. plug in 2. turn on light 3. adjust lamp brightness to medium ( mid range) 4. look into eyepieces 5. 10 x objective (yellow) - 4x useless because only 40x magnification 6. close iris diaphragm to 10 x ( to the right) 7. set inter-pupillary distance. by moving the eyepieces in and out until you see one circle and not two overlapping circles 8. put slide on stage with clip 9. center specimen over light coming through condenser 10. put 10 x objective ( yellow); use coarse and fine focus to focus parfocal-can switch between Objective lenses without significant refocusing. *You do not need to move the stage out of the way when you switch from one Objective lens to another. -auto stop

Antibiotic Sensitivity Testing - Optochin

Streptococcus pneumoniae is the only species in the genus Streptococcus that is susceptible/sensitive to the effects of Optochin. It will not grow around the disc. All other Streptococcus spp. are resistant to Optochin. Optochin has no effect on their growth. When you have a Streptococcus spp., Optochin Sensitivity is a Presumptive Test for Streptococcus pneumoniae. That means when you have a Gram Pos. Coccus that is Catalase Neg. and does not grow on Bile Esculin Agar, the Bacitracin Sensitivity test result allows you to determine if the organism in question is Streptococcus pneumoniae or not. Sensitive/Susceptible = Streptococcus pneumoniae Resistant = Not Streptococcus pneumoniae The Optochin Sensitivity Test is a Presumptive Test for the identification of Streptococcus pneumoniae. Because Streptococcus pneumoniae is susceptible (sensitive) to Optochin, there will be a zone of inhibition of growth around the disc . All other Streptococcus spp. are resistant to Optochin. Optochin has no effect on their growth . Since we do not have strain of Streptococcus pneumoniae that is safe for us to use in this laboratory, you will not actually see an organism that is sensitive to Optochin in this class

CAMP Test

Purpose: To determine an organism's ability to produce and elaborate CAMP Factor. CAMP Factor acts synergistically with staphylococcal β-hemolysin (β-lysin) on sheep erythrocytes to produce a lytic phenomenon when the two organisms grow near each other on agar containing sheep red blood cells. The incomplete (α ') hemolysis in the outer zone surrounding the Staphylococcus aureus will become complete (β) hemolysis in an arrow-head shape. The CAMP Test is a Presumptive Test for Streptococcus agalactiae. So, when you have a Gram Pos. Coccus that is Catalase Neg. and does not grow on Bile Esculin Agar, the CAMP Test allows you to determine if the organism is Streptococcus agalactiae or not. CAMP Test Pos. = Streptococcus agalactiae CAMP Test Neg. = Not Streptococcus agalactiae Bile Esculin Agar If Bile Salts inhibit the growth of the organism, the medium will be unchanged, with no colonies on the surface (the majority of Streptococcus spp.). If the organism can grow in the presence of Bile Salts, you should see growth on the surface of the medium (often tiny colonies). If the organism can break down esculin, the medium will turn black. You may notsee obvious growth on the surface, but if the medium has changed color, the organism must have grown. Streak Staphylococcus aureus one time across the center of a sheep blood agar plate. Don't go back and forth, just once across the center of the plate. Make 2 or more streaks of the organism to be tested parallel to one another, but at right angles to the Staphylococcus aureus. You want these streaks to be at least 1cm apart. Do not touch the Staphylococcus aureus streak when you put the suspected Streptococcus sp. organism on the plate. Incubate 37 °C for 24-48h For today, use one blood agar plate to compare Streptococcus pyogenes and Streptococcus agalactiae The CAMP Test is a Presumptive Test for the identification of Streptococcus agalactiae. Streptococcus agalactiae is the only species in the genus Streptococcus that produces CAMP Factor. CAMP Factor enhances the hemolysis in the outer zone of hemolysis around Staphylococcus aureus turning the hemolysis from incomplete to complete. The area of enhanced hemolysis is usually roughly arrowhead shaped and points toward the streak of Staphylococcus aureus. The CAMP test can also be used in the identification of Listeria monocytogenes (a Gram positive bacillus) Plate A shows a Streptococcus sp, with a Negative CAMP Test result. The organism is not Streptococcus agalactiae. - Plate B shows a Streptococcus sp, with a Positive CAMP Test result. The organism is Streptococcus agalactiae

Coagulase Test

Purpose: To determine whether the suspected Staphylococcus sp. under test can produce coagulase. Coagulase: Not actually an enzyme. (Thought it was when it was discovered. Oops. Too late. It already had an accepted name. Deal.) An extracellular protein that triggers coagulation of Rabbit plasma. The organism in question is inoculated with a loop into 0.5 ml of rabbit plasma and incubated at 37 ºC for 24 to 48 hr. Note: Because the Rabbit plasma is in a lightweight plastic tube, do not attempt to sterilize the top of the tube in front of the incinerator. A positive test is denoted by the formation of a clot in the test tube after the allotted time (i.e. the plasma gels up). Does the organism cause rabbit plasma to gel up? Does it produce coagulase, which triggers coagulation? Be sure the cap is snapped firmly onto the top of the tube and tip the tube. Coagulase Positive Staphylococcus spp. tend to be potential pathogens (causes of disease). Coagulase Negative Staphylococcus spp. tend to be non-pathogenic (harmless).

Antibiotic Sensitivity Testing - Bacitracin

Streptococcus pyogenes is the only species in the genus Streptococcus that is susceptible/sensitive to the effects of Bacitracin. It will not grow around the disc. All other Streptococcus spp. are resistant to Bacitracin. Bacitracin has little to no effect on their growth. When you have a Streptococcus spp., Bacitracin Sensitivity is a Presumptive Test for Streptococcus pyogenes. That means when you have a Gram Pos. Coccus that is Catalase Neg. and does not grow on Bile Esculin Agar, the Bacitracin Sensitivity test result allows you to determine if the organism is question is Streptococcus pyogenes or not. Sensitive/Susceptible = Streptococcus pyogenes Resistant = Not Streptococcus pyogenes The Bacitracin Sensitivity Test is a presumptive test for the identification of Streptococcus pyogenes. Because Streptococcus pyogenes is susceptible (sensitive) to the effects of Bacitracin, there will be a zone of inhibition of growth around the disc. All other Streptococcus spp. are resistant to Bacitracin. Bacitracin has no effect on their growth . It may help to look at your plate from underneath. Streptococcus pyogenes produces complete hemolysis. Since it can't grow under and around the Bacitracin disc, there will be a circle where no hemolysis has occurred under the Bacitracin disc - the medium is still red because the rbc's are still intac

RapID

** listen to link For example: RapID One for ident. of Enterobacteriaceae. RapID NF Plus API for ident. of oxidase + Gram - bacilli, including Vibrio spp. RapID ANA II System for ident. of anaerobes. RapID YEAST Plus System for ident. of yeasts. RapID STR for ident. of Streptococcus spp. RapID CB PLUS System for ident. of Corynebacterium spp. and other Gram + coryneform bacilli. RapID SS/u System for ident. of common urinary tract pathogens. There are test strips designed to identify a wide variety of organisms. RapID NH System for ident. of Neisseria, Moraxella, Haemophilus and related microorganisms. RapID STAPH PLUS System for ident. of staphylococci and related genera. Biomerieux produces over 20 different Analytical Profile Index (API) strips for identification of bacteria and yeasts. Becton Dickinson produces a number of tube style test systems.

The Brewer Jar and the Gas-Pak System

A Brewer Jar is an airtight chamber that allows us to incubate cultures of microorganisms under anaerobic conditions without the need for expensive equipment. The Gas-Pak System uses a sachet of chemicals that is activated when the foil packet is opened. Upon exposure to O2, a chemical reaction is initiated producing H2 and CO2. A catalyst in the sachet speeds the reaction of H2 with O2 to form H2O. Many Obligate Anaerobes will grow better in a CO2 rich environment. Methylene Blue is used as a Redox indicator. Methylene Blue is blue in an aerobic atmosphere and turns colorless when O2 has been removed from the atmosphere (anaerobic atmosphere). After the cultures have been inoculated, they are placed in the Brewer Jar along with an opened Gas-Pak sachet, and an indicator strip. The lid of the jar is sealed and the Gas-Pak removes the O2 from the atmosphere inside the jar. As anaerobic conditions become established, the indicator strip changes from blue to white (the Methylene Blue becomes colorless). The jar is incubated at 37C for 24 to 48 hours. After incubation, before the jar is opened, the indicator strip is observed. If the indicator strip is white, you can safely assume that the atmosphere inside the jar is anaerobic. If the indicator strip is blue, there is O2 inside the jar and you can not assume that the cultures were incubated under anaerobic conditions.

Enzyme Linked Immunosorbent Assay

A diagnostic test using Antigen-Antibody reactions Remember the basic structure of an antibody (we will limit ourselves to Immunoglobulin G, IgG). • The variable region (Fab) contains 2 antigen binding sites that bind specifically to one type of antigen. • The constant region (Fc) is the same for all IgG molecules from the species that produced the antibodies. For example, all human IgG molecules have essentially identical Fc regions. An enzyme linked antibody has an enzyme covalently bonded to the Fc region of the Immunoglobulin molecule. An ELISA can be used to detect Antigens in a sample (Antigen Capture ELISA) or to detect Antibodies in a sample (Antibody Capture ELISA). During the early phase of infection, Antigens from the pathogen are more abundant. During the later phase, the body will usually produce Antibodies and the amount of Antigens will decrease. After recovery from the infection the amount Antibodies rises even higher There are a number of factors involved, but for some diseases it is easier to develop an ELISA to detect the Antigens from the pathogen. For other diseases it is easier to detect Antibodies to the Pathogen. At the pharmacy you can purchase ELISA kits to detect various illicit drugs, or the hormones of pregnancy (pregnancy tests). We use Positive and Negative Controls to confirm that the assay was performed correctly. If the controls work, you can trust the results. Many ELISA kits are set up in triplicate. The 3 Positive control wells should all yield positive results. The 3 Negative control wells should all yield negative results. Ideally all 3 wells for each sample will agree (all +, or all -). If not, the 2 wells that agree are taken as the correct result. ELISA is commonly performed using a plate with 96 wells. If you have the money for fancy machines you can use a 384 well plate and let the robot do the work. We use a 12 well strip in a frame in order to avoid wasting the rest of a plate.

Remel RapID One

A set of 19 tests in a strip Plus an Oxidase Test. Each person gets their own personal unknown in a pure culture. You may pretend that you received a sample from a clinician, streaked a plate and obtained a pure culture. What is a McFarland Standard? Originally, a very carefully measured amount of barium chloride in sulfuric acid that would result in precipitation of barium sulfate. We now use McFarland Latex Standards. Very small latex particles in a buffer solution. The turbidity (cloudiness) of a McFarland Standard corresponds to a concentration of bacteria in suspension. The turbidity of a 0.5 McFarland Latex Standard is comparable to a bacterial suspension of 1.5 X 108 CFU/ml Prepare a suspension of your unknown organism in saline. Using a sterile swab, transfer 3-4 colonies to the pre-measured tube of Remel RapID Inoculation Fluid and screw the cap back on. Compare your suspension to a McFarland Standard of 2. ֎ Shake the McFarland Standard and your suspension well before comparing in front of a white card with black lines. You want your suspension to have the same turbidity (cloudiness) as the McFarland Standard. If you run this test in the real world, you want to match the turbidity of the McFarland Standard as closely as possible. Slightly more turbid is better than less turbid. For this exercise, slightly more or less turbid than the McFarland Standard is OK.

Streptocard

Antibodies to Streptococcal group specific antigens can be produced by injecting an animal with a sample of the purified antigens. • Those antibodies can be used in diagnostic testing. • The Streptocard Test Kit uses microscopic latex beads coated with rabbit antibodies that bind to Streptococcal group specific antigens. • Anti-A antiserum contains latex beads coated with antibodies to Group A antigens, Anti-B antiserum has antibodies to Group B antigens and so on. The appropriate antiserum will cause agglutination (clumping) when mixed with a sample containing antigens extracted from a Streptococcus sp. of the matching group. • When the clumps get large enough, they become visible to the naked eye. • No agglutination will occur if the sample does not contain homologous antigens (antigens that match the antibodies). • A fresh (18 to 24 hour) culture of a Gram Positive Coccus that is Catalase Negative is used in the Streptocard test. • So, when you have a pure culture of a Gram +, Catalase - Coccus, you could use this test to determine if the organism is a pathogenic Streptococcus sp. • In that case, you may not go through the process of identifying the organism by biochemical tests the way we have discussed for most of the semester. • A diagnostic laboratory might run additional tests to determine the susceptibility or resistance of the organism to various antimicrobial drugs. Kirby-Bauer Testing is one way to determine that. Each student has a Streptococcus sp. to test and a tube containing 400ul of BD BBL Streptocard extraction enzyme in a buffer solution. •Using an inoculating loop, transfer 4 colonies of your organism into the extraction buffer tube. • Label your tube with your seat number and put it in the 37oC dry bath for 10 minutes. (A dry bath is a block of aluminum on top of a heat source that holds a set temperature.) • Retrieve your tube and take it back to your seat. • Label the circles on your Streptocard test card A, B, C, D, F, & G. • Take your Streptocard Test card to one of the TA's in the middle of the room. • A TA will dispense Antisera to Streptococcal groups A, B, C, D, F, & G. • Back at your seat, use a transfer pipet to put one (just one) drop of your extracted antigens in each circle Using a new stick for each circle, gently mix the antigen extract and antiserum to fill the circle. • Put each stick into a red biohazard bag immediately after you use it. Don't just lay it down somewhere. • After you have mixed all the circles, gently rock the card with a circular motion for 30 seconds to 1 minute. Your TA's would love to demonstrate if necessary. • You should start to see agglutination in one of the circles after about 30 seconds.

Aerotolerance Testing: some background

Cells need to repackage energy form their food source(s) into amounts that are useable in cellular metabolic processes. Respiration is a bio-oxidative process in which O2, or some other inorganic compound, is used as the final electron acceptor in the Electron Transport System. • In Aerobic Respiration O2 serves as the final electron acceptor. • In Anaerobic Respiration an inorganic molecule such as Sulfate (SO4 - ) or Nitrate (NO3 - ) serves as the final electron acceptor in place of O2. • Anaerobic Respiration is not a significant factor when identifying medically important bacteria, so that is all we will say about it in lab for this course. Fermentation is a bio-oxidative process in which an organic compound serves as the final electron acceptor in the Electron Transport System. The enzymes used for fermentation do not require or use O2. • In respiration, organic molecules are degraded completely to inorganic compounds. Degradation of glucose by Respiration will produce approximately 38 ATP's per glucose molecule. • In fermentation, organic molecules are not degraded completely. Organic compounds are produced by fermentation. Degradation of glucose by Fermentation produces approximately 4 ATP's per glucose molecule. • Respiration produces more energy from a glucose molecule than fermentation. In an O2 rich environment (normal room air atmospheric conditions i.e. 20% O2) there will be highly reactive oxygen species present. Highly reactive O2 species are very chemically reactive forms of Oxygen. They include: Superoxide O2 .- (an O2 molecule with an extra electron) Hydroxyl radicals OHHydrogen Peroxide H2O2 In order to be able to grow in the presence of O2, an organism must have enzymes that allow it to detoxify the highly reactive O2 present in its environment. Organisms that lack enzymes to detoxify highly reactive O2 species cannot grow under normal room air atmospheric conditions. Superoxide radicals (O2 -) • Are formed during the incomplete reduction of oxygen during electron transport in aerobes and during metabolism by anaerobes in the presence of oxygen. They are detoxified by superoxide dismutase. 2O2 - + 2H+ H2O2 + O2 • Peroxide anion (O2 -) • Is a component of hydrogen peroxide, which is formed during reactions catalyzed by superoxide dismutase. The enzymes catalase and peroxidase detoxify peroxide anion. • 2H2O2 H2O + O2 • Hydroxyl radicals (OH·) • Result from ionizing radiation and from the incomplete reduction of hydrogen peroxide. Hydroxyl radicals are the most reactive of the toxic forms of oxygen, but because hydrogen peroxide does not accumulate in aerobic cells, the threat of hydroxyl radicals is virtually eliminated in aerobic cells. procedure 1. divide plate into thirds 2. one incubated in brewer jar or room temp 3.packet- O2 bound to chemicals and O2 bound too N2 and packet generated H2 and CO2; takes O2 out of atmosphere in jar 4. put cultures in jar and open packet, indicator strip add ( methylene blue redox indicator), seal jar - strip turns white= anaerobic 5. sterilize the loop, pick up colony, streak plate in organism segment on plate, resterilze loop 6. pick up other colony, streak on plate 2 7. repeat with other 2 ( one in brewer jar because would die out in open) organisms and streak on both "anaerobic and aerobic plate", resterilize loop 8. place organism back in anaerobic. condition; put one of two plates in brewer jar, add. indicator strip, open packet and put in jar, seal jar ( gen H2. CO2 byproduct, cat speed up rxn. of H2O and. remove. O2 out. of. atmosphere), incubate one in room air and brewer jar

Fluid Thioglycollate Medium

Fluid Thioglycollate Medium is a semisolid medium that contains Sodium Thioglycollate, a reducing agent, that binds O2 in the medium and creates a relatively anaerobic environment in the bottom portion of the tube. The medium (the formulation we use) also contains a Redox (OxidationReduction) Indicator that turns blue-green in the presence of O2. Some labs use Resazurin as a Redox Indicator. Resazurin turns pink/red in the presence of O2. I am attempting to produce or find high quality images to show you When the tube is heated with cap loose, O2 is driven out of the medium. When the tube has cooled enough to handle, the cap is tightened. This prevents additional O2 from entering the tube. Some of the O2 from the atmosphere inside the tube diffuses into the medium. The Redox Indicator shows that near the surface, O2 is present in the medium. Thioglycollate binds O2 so that as the gas diffuses into the medium, it gets trapped. Only the medium near the surface will be oxygenated (the Oxic zone). The medium toward the bottom of the tube remains anaerobic (the Anoxic zone). The medium is inoculated by stabbing to the bottom of the tube. Ideally, a needle is used, but since the medium is only semi-solid, we use a loop. Any gap produced seals back up. There won't be any air bubbles trapped in the medium. The tube is incubated for 24 to 48 hours at 37C. Tube a: Growth has occurred only in the Oxic zone, indicating that the organism requires O2 for growth. Obligate Aerobe. Tube b: Growth has occurred only in the Anoxic zone, indicating the organism can only grow under anaerobic conditions. Obligate Anaerobe. Tube c: Growth has occurred throughout the tube, but the organism has grown better with O2 and more slowly without O2. Facultative Anaerobe. Tube d: Growth has occurred almost exclusively at the interface between the Oxic zone and the Anoxic zone, indicating that the organism requires O2 for growth, but cannot tolerate a normal atmospheric level of O2. Microaerophile. Tube e: Growth has occurred evenly throughout the tube. The growth rate was the same in both the Oxic zone and the Anoxic zone. The concentration of O2 has no effect on the growth of the organism, indicating the organism does not require O2 for growth, but is not inhibited by the presence of O2. Aerotolerant Anaerobe.

Antibody Capture ELISA

For this exercise we will use an ELISA to look for Antibodies to, let's say influenza. Coat all the wells with Antigens from the pathogen. Protein will stick to plastic. Wash the wells twice with Wash Buffer to remove unbound antigens. The Wash Buffer will also coat any plastic surfaces that remain exposed. This will prevent other proteins (like random IgG molecules in your sample) from sticking to the wells Add your sample. If the sample contains antibody specific for the antigen (primary antibody), it will bind. Antibodies to other antigens will not bind. • Antigen-Antibody binding is extraordinarily specific. Wash the wells twice to remove any unbound antibodies Add Enzyme linked Antibody (Secondary Antibody). If Primary Antibody has bound to the Antigen, 2o Antibody will bind to the 1o Antibody. Wash the wells twice with Wash Buffer to remove unbound 2o Antibody. Add the substrate (which is colorless). If the enzyme is present, it will act on the substrate and the well will turn blue after a short incubation. The enzyme linked antibodies (the 2o Ab's) are specific for the Fc portion of the 1o antibodies you are trying to detect. In our example, we are trying to detect human IgG antibodies. To produce the 2o antibodies: We would inject, let's use a goat, with human IgG. The goat will produce antibodies to the foreign proteins (human IgG). We collect the goat Ab's, purify them, and label them with our enzyme, and we have our Enzyme-linked 2o Ab's.

When you are in a Medical Microbiology Laboratory, You know you have a (insert genus here) when...

If the organism: Is a Gram Positive Coccus Is Catalase Positive Grows on Mannitol Salt Agar It is a Staphylococcus species. If the organism: Is a Gram Positive Coccus Is Catalase Positive Cannot grow on Mannitol Salt Agar It is a Micrococcus species. If the organism: Is a Gram Positive Coccus Is Catalase Negative. Cannot grow on Bile Esculin Agar It is a Streptococcus species. If the organism: Is a Gram Positive Coccus Is Catalase Negative. Grows on Bile Esculin Agar It is an Enterococcus species or Streptococcus bovis.

RapID One Interpretation

If you have someone else's RapID ONE strip in front of you, tell us now. You need to add reagents to some of the wells, but pay attention to which wells and when. Those who don't listen tend to get bad results You need to add reagents to some of the wells, but pay attention to which wells and when. Add 2 drops Remel RapID ONE reagent to the wells/cavities labelled PRO, GGT, and PYR. ֎ The maroon arrow indicates a little pull tab. ֍ The maroon box is my addition to highlight the wells of interest. Read all the wells from URE to IND (1 - 18) and record the results on the report form. ֎ At this point: Some of you are asking, "How do I read the results and how do I record them?" Some of you are sleeping. Don't wake them. Just laugh softly when they seem confused in a few minutes. Some of you may have already attempted to get ahead of everyone else and messed up beyond repair. To read the results, compare the colors in the wells to the color guide provided to you. I suggest putting your strip on top the chart to compare the wells to corresponding circles. Record the results on the report form. The rows for 'Value' and 'Result' are highlighted at right. For negative results, put a zero (0) in the result box. For positive results, write the number from the 'Value' row in the result box (1, 2, or 4). You may have noticed that the result boxes for PRO (Test 15), GGT (Test 16), PYR (Test 17), and IND (Test 19) are shaded. That's because you need to add the appropriate reagent to each of those wells before you read those tests. ֎ Be sure you read and record the result for ADON (Test 18) before you add the reagent for IND (Test 19). ֎ This is where somebody messes up every semester. Don't be 'That student'. The giggling of your neighbors will be difficult to forget. You may have noticed that the result box for OX has a '-' in it already. The RapID ONE strip is designed for identification of organisms in the family Enterobacteriaceae. They are all Oxidase neg. Now add 2 drops of RapID Spot Indole Reagent to the well/cavity labelled ADON/IND. Record the result for IND. You may have noticed that the 'Value Total' row has the tests separated into groups of 3 tests. • A positive result for the first test of each group gets a value of 1. • A positive result for the second test of each group gets a value of 2. • A positive result for the third test of each group gets a value of 4. • Remember, all negative results get a value of 0. We add the result values in groups of 3. • Consider that this system gives us a value total for each group of 3 tests from 0 to 7. • Each value total from 0 to 7 corresponds to a different combination of positive and negative results. • +, +, - gives a value total of 3. • -, +, + gives a value total of 6. At this point you will have a 7-digit code that we will enter into the on-line computerized database. It's name is Eric.The database will spit back an identification We would like to see a result that says Probability level: Implicit. -Probability level: Adequate or Satisfactory is nice (we sometimes see these) -Since we are using Laboratory adapted strains, it is not unusual to see Probability Level: Unacceptable. That means your code does not match anything the computer can identify. We will take a look at your strip to see if we can reinterpret the colors to make Eric happy. Remember that we are using Laboratory adapted strains and the database contains clinical strain Write the identification of your unknown (from Eric) on your report form. If your TA or Laboratory Instructor tells you it was something else, put that on the form too and make a note about what you got vs. what we said we gave you. Names have been changed to protect the Instructor.

Kirby-Bauer Antimicrobial Susceptibility Testing pt.2

Measure, in mm, the diameter of each zone of inhibition. If the bacteria grew up to the edge of the disc, record the diameter as 0mm and conclude that the organism is resistant to the drug in the disc. Compare your measurements to a chart to determine if the organism shows clinical susceptibility, intermediate susceptibility, or resistance to the drug. Measure, in mm, the diameter of each zone of inhibition. If there are colonies inside the zone of inhibition, measure from the center of the disc to the inner edge of the closest colony and double that measurement. You don't want to select for resistance when you give an antimicrobial drug. In the image to the right, you would record a diameter of 0mm for C 30. Keep in mind that if there is a zone of inhibition of the organism around a disc, the only way to determine the level of clinical susceptibility is to measure the diameter and check a chart. A bunch of people worked very hard to measure the concentration of each drug at various distances from the discs and correlate those concentrations to the concentrations in a patient's body. If the diameter you measure for a drug falls in the resistant range, you can't give a patient enough of the drug to achieve effective treatment. When 2 drugs have Zones of Inhibition of different diameters, you cannot simply assume the larger diameter zone of inhibition means greater susceptibility. • Different drugs will diffuse through the Mueller-Hinton Agar at different rates, and the discs may have different amounts of the drugs Comparing zones of inhibition: Since there is no inhibition of this organism resulting from disc B, you can safely say the organism is resistant to the drug. Depending on what drugs were used in this test, the organism may actually be susceptible to the drug in discs C and D, but resistant to the drugs in discs A and E The results of the Kirby-Bauer Antimicrobial Susceptibility Test are only part of the information necessary to choose an appropriate antimicrobial drug for the treatment of an infection. Other factors to consider in choosing an antimicrobial drug include: The location of the infection. e.g. Urinary tract infection - Some drugs pass through the kidneys into the urine in an active form, some won't reach the urine in an active form. The desired route of administration. Some drugs can be given orally. Some are only effective when given intravenously. Some drugs are only used topically. The patient may be allergic to certain drugs. Dr. Turner had an allergic reaction to penicillin when he was a child. Do not give Dr. Turner Penicillin (unless, of course, you would prefer a different Laboratory Instructor). Other factors to consider include (cont'd) Pre-existing health conditions of the patient. Some drugs can be nephrotoxic, so they should not be given to a patient with kidney disease. Species of the patient: Chloramphenicol can cause aplastic anemia in some people. It is illegal to give Chloramphenicol to a food animal due to the potential for residual amounts of the drug in products coming from that animal (meat, milk, eggs). If you give penicillin to a guinea pig it will die. Penicillin will disrupt it's normal gastrointestinal flora.

RapID One Inoculation

Peel back the corner (you may refer to the image below) of the cover and, using a transfer pipet, transfer the entire volume of bacterial suspension to the test strip. Peel back the corner (you may refer to the image below) of the cover and, using a transfer pipet, transfer the entire volume of bacterial suspension to the test strip. Tip the strip back to a 45o angle and rock the strip side to side to evenly distribute the saline suspension. Check to see that the suspension is evenly distributed across the strip. Gently rock the strip forward until the bacterial suspension runs into the wells. Tip the strip back to a 45o angle (Fig. 1) and keeping the strip tipped back, rock the strip side to side (Fig. 2) to evenly distribute the saline suspension. Check to see that the suspension is evenly distributed across the strip. Gently rock the strip forward until the bacterial suspension runs into the wells Put your initials and seat number on the strip. Put the strip in incubation tray in the middle of the lab (section # and date are nice, but optional). We will incubate the strips for 4 hours at 37C and then put them in the refrigerator until Thursday. ֍ In the real world you would read the results after 4 hours of incubation (this is a 1 hour lab)

Setting up the Kirby-Bauer Test

Prepare a suspension of your unknown organism in saline. Using a sterile loop, transfer 4 - 5 colonies of the organism to the screw-cap tube of saline and screw the cap back on. Compare your suspension to a MacFarland Standard of 0.5. Shake the MacFarland Standard and your suspension well before comparing in front of a white card with black lines. Never open the MacFarland Standard tubes. They are very expensive. That's why they are taped closed. Prepare a suspension of your unknown organism in saline (cont'd). You want your suspension to have the same turbidity (cloudiness) as the MacFarland Standard. If you run this test in the real world, you want to match the turbidity of the MacFarland Standard. The life of a patient could depend on the lab getting the correct answer. With practice, you get an idea of how many colonies you will need to get the right turbidity. For this exercise, slightly more or less turbid than the MacFarland Standard is OK. Dip a sterile swab into the saline suspension and swab the ENTIRE surface of the plate in 3 different directions. You want to create a "lawn" of bacteria on the plate • Give your plate a few minutes to dry (with the cover on the plate). • When it is dry take it to the back of the lab and one of the TA's will show you how to dispense the antimicrobial impregnated discs. • Work on one of the solid lab tables, not the folding tables. The folding tables are bouncy. • You should end up with 12 discs on your plate. • If the dispenser jams or won't dispense discs, STOP USING IT IMMEDIATELY, IT PROBABLY NEEDS TO BE REFILLED. • Before you turn your Mueller-Hinton Agar plate over for incubation, use a sterile toothpick to tap each disc lightly to ensure it stays stuck to the agar surface. • Label your Mueller-Hinton Agar plate with your table and seat #, and put it in the tub in the middle of the lab. • We will move them to the refrigerator after 24 hrs. of incubation.

Novobiocin Sensitivity Test

Principle: Organisms that are susceptible/sensitive to a given antimicrobial compound will be inhibited from growth in the presence of that compound. Biochemistry Involved: Novobiocin is an antibiotic obtained from Streptomyces niveus and other Streptomyces spp. It is an inhibitor of prokaryotic DNA gyrase, and effective chiefly against some Staphylococcus spp. The Novobiocin Sensitivity Test is a Presumptive Test for Staphylococcus saprophyticus. If you have a Gram Pos. Coccus that is Catalase Pos. and it grows on Mannitol Salt Agar, it is a Staphylococcus sp. A Staphylococcus sp. that is resistant to Novobiocin is Staphylococcus saprophyticus. Staphylococcus saprophyticus is the only Staphylococcus sp. that is resistant to Novobiocin. A Staphylococcus sp. that is resistant to Novobiocin is Staphylococcus saprophyticus. A Staphylococcus sp. that is sensitive to Novobiocin is NOT Staphylococcus saprophyticus. Both of these organisms are Staphylococcus spp. They are Gram Pos. cocci They are Catalase Pos. They grow on Mannitol Salt Agar. Novobiocin inhibits the growth of the Staphylococcus sp. in the top image. It is sensitive to Novobiocin. It is not Staphylococcus saprophyticus. Novobiocin has no effect on the growth of the Staphylococcus sp. in the bottom image. It resistant to Novobiocin. It is Staphylococcus saprophyticus.

Lab 7 prep

RapID strep - Mcfarland standard of 2; turbid and not clear like inoculation tube because no bacteria in. there 1. enterobacterace organism, sterilize, pick up 5-6 colonies, put in inoculation fluid after sterilize tube, sterilize, cap, sterilize loop, label strip 2. shape to get in suspension 3. hold with test card next to mcfarland standard 4. two tubes are of similar turbidity we have appropriate. concentration in inoculum fluid 5. pipet, open strip in corner, grab incoulation fluid with pipet and put on strip ( repeat 3 times until all incoluation fluid on strip) 6. dispose of pipet 7. tip tray back and rock back and forth to make sure evenly. distributed, rock forward and make sure fluid goes in tube; level of suspension should be same in. all wells Kirby Bauer- what. drugs to use 1. label plate, have saline, use mcfarland standard of .5 2. sterilize loop, dont need as many colonies so pick up 2-3 colonies, place in incoulation tube after sterilizing top, sterilize loop and tube, mix tube 3. hold next to card with standard and make sure they are the same 4. put swap in suspension of bacteria, sterilize tube each time, streak entire plate across ( 3 angles), swab in biohazard bag 5. put 12 disk with dispensor all over plate 6. get toothpick and tap each disk down to make sure flat on surface. of medium 7. turn over so they hang off agar and incubate *keep in jar so the disks dont. absorb moisture aerotolerance test 1. 3 tubes of TGH medium, dont shake, top. 2 cm are pink (rhizarium) and bottom is yellow ( differences in oxygen zones), label tubes 2. sterilize loop, pick up colony, open TGH tube and put loop in and pull out, sterilize, recap tube 3. repeat for other 3 samples aerotolerance test on plates with anaerobe jar 1. label plates; 2 plates per organism 2. sterilize loop, grab colony, streak for isolation on. plate, sterilize loop between. each streak and after 3. repeat on each plate per organism 4. incubate one. of. each in anaerobic jar/ brewer jar with redox indicator strip & packet, and other 3 in normal air -strip from blue-> white to indicate anaerobic enviornment

Bile Esculin Agar

Selective/Differential Medium - used to differentiate Enterococcus spp. and Streptococcus bovis from other Streptococcus spp. Also to differentiate organisms that can break down esculin from those that cannot Selective: • Bile Saltsinhibit non-enteric organisms. Differential: • Esculin - potential substrate - Some organisms can split esculin to form esculetin (byproduct) and glucose (food source). • Iron - Indicator. - Reacts with esculetin to form a black precipitate.

Mannitol Salt Agar

Selective/Differential medium - used to select for halophilic organisms (primarily Staphylococcus spp.) and differentiate mannitol users from non-mannitol users. Selective: • High salt concentration (at least 7.5% NaCl) prevents the growth of non-Halophilic organisms. • Halophilic organisms grow with no problem. Differential: • Mannitol - Potentialsubstrate • Phenol Red - pH Indicator Most organisms are not Halophilic and cannot grow. Halophilic bacteria grow (among the medically important Gram + cocci, Staphylococcusspp.) Bacteria utilizing the Mannitol will result in ↓ pH, the phenol red will turn from pink (Neutral) to yellow (acid). Bacteria NOT utilizing the Mannitol will result in ↑ pH, the phenol red will turn from pink (Neutral) to Bright pink (alkaline).

The Aerotolerance Test

The Aerotolerance Test is a way to determine the O2 requirements of one or more organisms. For this test, a medium that contains a reducing agent, such as CDC Blood Agar, is generally used. A reducing agent will bind O2 and help ensure the bacteria will be held under anaerobic conditions when placed in an anaerobic environment. Always label your culture plate appropriately. We will inoculate each of 2 CDC Blood Agar plates with 4 bacteria as indicated: • Escherichia coli (E. coli) • Micrococcus luteus • Bacillus cereus • Clostridium sporogenes One plate will be incubated under normal room air conditions. The other plate will be placed in a Brewer Jar with a Gas-Pak. Both plates will be incubated at 37C until Thursday An Obligate Aerobe should show growth only on the plate incubated under room air conditions (20% O2). A Facultative Anaerobe should show growth on both plates as it can grow both with and without O2. We expect it to have grown better with O2 than without O2. An Obligate Anaerobe should show growth only under anaerobe conditions, so only on the plate incubated in the Brewer Jar The plate at the top was incubated under room air (aerobic) conditions. The plate at the bottom was incubated in a Brewer Jar under anaerobic conditions. Organism A grew only under aerobic conditions - Obligate Aerobe. Organism B grew only under anaerobic conditions - Obligate Anaerobe. Organisms C and D grew on both plates, but grew faster under aerobic conditions and slower under anaerobic conditions - Facultative Anaerobe. Your goal is to determine the O2 requirements of all 4 organisms. Had you been able to complete this exercise in the laboratory, most of you would have picked up on a subtle difference. You would have received Organisms A, C, and D on blood agar plates sitting on the table in front of you. You would have inoculated Organisms A, C, and D onto both test plates, then we would have given you Organism B. You would have been instructed to your plates with organism B and take one plate directly to your TA so that we could get Organism B back into anaerobic conditions before it died from exposure to atmospheric O2.

Oxidase Test

The Oxidase Test is a test for the enzyme Cytochrome c Oxidase. Some organisms use Cytochrome c Oxidase as the final enzyme in the Electron Transport Chain. Cytochrome c Oxidase catalyzes the oxidation of Cytochrome c and the reduction of O2 to form water. [That means, it transfers electrons (hydrogen ions, H+) from Cytochrome c to O2] The Oxidase Test reagent, tetra-methyl-p-phenylenediamine (Just call it Oxidase reagent for this class) can serve as an artificial electron donor for Cytochrome c Oxidase. When the Oxidase reagent is reduced, it changes from colorless to blue/purple. The Oxidase Test is primarily used in the Biomedical Microbiology laboratory to differentiate Gram negative bacilli in the family Enterobacteriaceae (which are oxidase negative) from Gram negative bacilli in the family Pseudomonadaceae (which are oxidase positive). It can also be helpful for distinguishing the Gram negative Neisseria gonorrhoeae (oxidase positive) from important Gram positive cocci, such as Staphylococcus spp. and Streptococcus spp. (oxidase negative). Procedure: Using clean, sterile toothpick for each organism to be tested, transfer a colony of the organism to a piece of filter paper. **In my experience, better results are obtained by scraping the sample into the filter paper for several seconds; irritating the snot out the bacteria until just before you tear a hole in the paper. I believe this breaks open many of the cells and exposes the enzyme. Many bacterial enzymes are bound to the inside of the cytoplasmic membrane. In this laboratory we use Pseudomonas aeruginosa as an oxidase positive control and Escherichia coli (E. coli) as an oxidase negative control. That means at least 3 smudges of bacteria - the test organism and 2 controls. Procedure: The oxidase reagent is supplied in a small dropper vial. • Never remove the top from the vial. • A vial of reagent holds 20 or 30 drops of reagent. **make sure not degraded • If the vial is new (unopened) you need to squeeze quite firmly to break the glass ampule inside the plastic tube. Point the dropper down toward one of your smudges of bacteria. • Never squeeze the vial with the top pointed up toward your face. Procedure: Put a drop of reagent on each bacterial sample on your filter paper. • You want to put reagent on your sample to be tested and the 2 control samples at the same time. • Don't contaminate the dropper by touching the bacteria samples. They have little hard hats on, the drop won't hurt them when it hits them. -After you add reagent, watch the paper until the test if finished. Don't go inoculating some other test, or reading your manual. The Oxidase reagent breaks down spontaneously when exposed to O2 in the air. If the reagent is fresh, Oxidase positive bacteria may take up to 1 or 2 minutes to turn the reagent blue. Smudges of Oxidase negative bacteria will probably begin to turn blue after 3 or 4 minutes, but may take much longer. If the vial has been open for a day or 2, Oxidase positive bacteria will likely begin to turn the reagent blue in 15 to 20 seconds. Smudges of Oxidase negative bacteria will likely begin to turn blue after as little as 30 seconds. I know what the manual says about the timing, and I don't care. Bacteria don't read lab manuals. Since the Oxidase reagent will turn blue by itself, you need to watch the test until either: • The smudge of known oxidase negative bacteria begins to turn blue, • The paper around the area that was wet with the reagent begins to turn blue, or • You have been watching the test for at least 1 minute after the oxidase positive control has turned a very convincing blue and no other changes have occurred. 1. label filter paper ( ox pos/neg control and smaples) all 4 on one filter paper 2. toothpick on colony scrape 3. smudge on paper ( aggrivate snot before think tear paper); oxidase positive will give better results 4.dont point towards face or take off top of ampule; squeeze hard; drop on samples 5. if dont work; check with ox pos 6. if doesnt work; put reagent on paper first and if is blue then it is old so need new tube; ox neg wont turn blue or take longer because reagent breaks down in air and turns blue and then youll have to restart (+)= blue (-)+ no color

Antimicrobial Sensitivity Testing

The terms Antimicrobial drug and Antibiotic are often treated as synonyms, but technically they are not. An Antimicrobial drug is a chemical compound that inhibits the growth of one or more microbes (bacteria, yeast, virus). An Antibiotic is an antimicrobial drug that is produced by a living organism. i.e. Penicillin Some Antimicrobial drugs are synthetic (produced in a Chemistry laboratory). i.e. Sulfa drugs (Sulfamethazine Sensitivity and Susceptibility are used synonymously. If a drug kills or inhibits the growth of an organism, the organism is Sensitive or Susceptible to the effects of the drug. A Bacteriostatic drug inhibits the growth of susceptible bacteria, but is not lethal to the organism(s). If the drug is removed from the environment, the bacteria remain viable . A Bacteriocidal drug kills susceptible bacteria. Fungistatic, Fungicidal, and Virucidal are related terms with similar meaning Purpose: To determine an organism's susceptibility to a specific antibiotic. We usually use blood agar for these tests. Using a sterile swab, pick up 3 to 4 colonies of the organism to be tested and swab to cover the entire surface of the plate. Use the same side of the swab to pick up your inoculum and streak the plate. Swab across the entire surface of the plate. You want bacteria everywhere across the entire surface of the medium Rotate the plate 90º and swab the plate so as to get a solid blanket using your cotton swab. You are attempting to cover the surface of the plate with the organism. You want any area of no growth to be the result of exposure to the Antibiotic, not simply a lack of bacteria from the beginning. Place an antibiotic disc on the plate near the center of the plate. Incubate at 37C until the next lab period. In a diagnostic laboratory, 24 hours In our laboratory: We test suspected Staphylococcus spp. for sensitivity (susceptibility) to Novobiocin. We test suspected Streptococcus spp. for sensitivity (susceptibility) to Bacitracin and Optochin. The Optochin and Novobiocin discs are in handy dandy little dispensers for you to use. The code for Optochin is 'P' because P is the second letter of the word. The code for Novobiocin is 'NB' just 'cause . Be careful not to shoot the disc across the table. The Bacitracin discs come in a little jar similar to the one seen here. The Bacitracin discs have an 'A' on them because, yes, that's correct, A is the second letter of the name. The TA's will dispense the Bacitracin discs with sterile forceps. They tend to stick together and we end up losing 6 or 7 extra discs per student if we just leave them out for everyone to get their own. For a suspected Staphylococcus sp., place a Novobiocin disk in the center of the plate For a suspected Streptococcus sp., place a Bacitracin disk on one half of the plate, and an Optochin disk on the other half of the plate. You can put both a Bacitracin disc and an Optochin disc on one plate of agar . Don't waste media. That is a silly and wholly unnecessary way to waste your tuition money

Computer-Assisted Multitest Microsystems

These systems combine multiple biochemical tests into a single strip. Designed for ease of inoculation and rapid identification of an unknown microorganism. Available from multiple manufacturers. Advantages over traditional culture tubes and plates include: Ease of inoculation Decreased time needed for set up. Require less incubator space. Use a computerized database to identify a wide variety of microorganisms. Less biohazard waste for disposal. The major disadvantage, cost of the strips (more expensive than traditional culture tubes and plates) is balanced by the reduction in other costs (i.e. technician time, more efficient use of incubator space, waste disposal)

Lab results

gram +; cat +->. strep or micro 1. mannitol salt agar; micro wont grow ( media is the same color as it started out, primary streak a little growth but cant grow isolated colonies because toxin/inhibited by media) 2. staph- grows, uses mannitol, isolated colonies from and makes media yellow turning the media acidic; mannitol + is pathogenic 3. grow but media is pink then uses peptones and doesnt use mannitol because media isnt yellow so non-pathogenic staph coagulase- gram + cat +; stph or micro; grew on mannitol salt so is staph 1. tip tube, coagulase + will gel up plasma at end of tube, if not then negative because plasma is still liquid * coagulase + staph= pathogenic and coagulase - staph= nonpathogenic novobiocin 1. inhibited there will be a zone of inhibiton and susceptible 2. no inhibition around then not susceptible *staph resistant to novobiocin- staph sepraciticus gram +, cat - -> entero or strep - bile eschuin agar 1. agar untouched-> strep ( color is pale yellow) 2. grows - enterococcus or strep bovus ( media is black) - reacts with iron-> tube black cAMP test 1. staph aureus on center of plate, maybe a strep species streak perpendicular to staph aureus; if there is enhancement of outer zone of hemolysis of staph aureus ( strep agalactea) ; where two organism near each other and no enhancement of zone of hemolysis gram +, cat -, bile - and suscept to optochin-> strep a galactea some red under disk-> inhibition of grow by bactiricin

Lab- inoculation/test prep 7/6

gram pos- catalase test 1. cat neg, pos control, and unknown. label on slide 2. clean toothpick, recap toothpick tubes, touch a colony and pick up, smudge onto slide 3. add drop of H2O2 onto colony 4. + will bubble - will not 5. slide disposal container *working with blood agar dont pick up blood agar because blood is cat + suscept to Bacitracin and optochin 1. clean swab, pick up colony and streak all over the plate, turning 3 times to get from 3 angles using same side of swab 2. repeat with other same 3. dispose of swabs 4. add Bacitracin and optochin disks to plate; use clean forceps to get bacitracin disk Novobiocin suscept 1.. clean swab, pick up colony and streak all over the plate, turning 3 times to get from 3 angles 2. repeat with other same 3. dispose of swabs 4. add novobiocin disk onto plate coagulase test 1. small tube. with rabbit plasma ( don't sterilize in front of incinerator because melt tube since plastic) 2. loop sterilize, grab colony, inoculate rabbit plasma tube wiggling, recap 4. resterilize loop and repeat with other culture cAMP test 1. sterilize loop or toothpick, pick. up staph aureus colony, draw one line across center of plate, straight line, resterilize 2. grab other samples and use loop, pick up colony, draw a. line from center line to edge of plate. for both *touch. source culture once or get a new loop/toothpick *space samples far enough apart mannitol salt agar 1. streak for isolation; see what colonies look like and do 2. loop sterilize, pick up colony, 4 streaks in labeled quadrants, the fourth streak streak across the center of plate, sterilize in between each streak 3. repeat with other two samples Bile Esculin Agar ( enter and strep bovus) 1. sterilize loop, pick. up colony, uncap agar tube, sterilize, streak surface as pull loop out,sterilize, cap, sterilize loop 2. repeat for other sample; keep cap a half turnloose so O2 can get in

Classification of microorganisms based on O2 requirements

• Obligate Aerobes Require O2 as the final electron acceptor in their electron transport chain. Cannot grow without O2 in their environment. Have enzymes to detoxify highly reactive O2 species. Use Respiration only. • Obligate Anaerobes Do not use O2 as the final electron acceptor in their electron transport chain. Cannot grow in the presence of O2. Do Not have enzymes to detoxify highly reactive O2 species. Use Fermentation only. Facultative Anaerobes Can use O2 or organic compounds as the final electron acceptor in their electron transport chain. Can grow with or without O2 in their environment. Have enzymes to detoxify highly reactive O2 species. Use Respiration when O2 is available, Fermentation when no O2 is available. Will grow more quickly when O2 is available, because Respiration yields a large amount of ATP. Will grow more slowly when no O2 is available, because Fermentation yields a small amount of ATP. • Aerotolerant Anaerobes Use only organic compounds as the final electron acceptor in their electron transport chain. Can grow with or without O2 in their environment. Have enzymes to detoxify highly reactive O2 species. Use only Fermentation. Will grow at the same rate with or without O2, since they always use fermentation to obtain energy from their food sources. • Microaerophiles Require O2 as the final electron acceptor in their electron transport chain. Cannot grow without O2 in their environment. Do Not have enzymes to detoxify highly reactive O2 species. Will only grow in an environment with a very low concentration of O2, less than normal atmospheric level. Use only Respiration . • Capnophiles Grow best in an atmosphere containing a high concentration of CO2 (perhaps 5%, normal atmospheric level is <1%) and lower than normal atmospheric level of O2.

Principle of Capsule Staining

• Capsules stain very poorly. because carbohydrate • A Negative stain can help with visualization of a capsule. ** things try to show in unstained therefore a negative stain • Negative staining methods contrast a translucent, darker colored, background with stained cells but an unstained capsule. The background is often stained with nigrosin. • A positive capsule stain requires a chemical that precipitates and stains the capsule. The cells and background are usually stained a darker color than the capsule • The capsule stain employs an acidic stain and a basic stain to detect capsule production. Capsules are formed by organisms such as Klebsiella pneumoniae. Most capsules are composed of polysaccharides, but some are composed of polypeptides. The capsule is a thick, detectable, discrete layer outside the cell wall. Capsules protect bacteria from the phagocytic action of leukocytes and allow pathogens to invade the body. Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces. Therefore, the best way to visualize them is to stain the background using an acidic stain and to stain the cell itself using a basic stain. We use India ink and Gram crystal violet. This leaves the capsule as a clear halo surrounding a purple cell in a field of black. The medium in which the culture is grown as well as the temperature at which it is grown and the age of the culture will affect capsule formation. Older cultures are more likely to exhibit capsule production. When performing a capsule stain on your unknown, be sure the culture you take your sample from is at least five days old. Procedure 1. A smear of bacteria is prepared at the center of the slide as follows: a drop of water is put at the center of the slide and a loop of bacteria from the plate or slant is transferred to it by a loop sterilized over flame. ** mucoid appearance Then, by slow rotation of the loop in the drop, a bacteria suspension is made and it is spread till a smear is obtained. 2. Using your inoculating loop, spread the sample out to cover about a 3/4 inch circle on the slide. 3. Let it completely air dry. Do not heat fix. Capsules stick well to glass, and heat may destroy the capsule. 4. Stain with crystal violet for one minute. 5. Wash off the excess dye with 20% copper sulfate solution. 6. Shake off the excess copper sulfate solution and immediately blot dry. 7. Observe using oil immersion microscopy. Observations: 1. Color of the area surrounding the cells: Light blue: Capsule present (capsulated bacteria) Dark purple-blue: Capsule absent (non-capsulated bacteria) 2. Color of the cells: dark purple-blue 3. If capsulated, size of the capsules: small, moderate or large. 4. Relative size of cells and capsules Procedure capsule stain- 1. drop of crystal violet on slide 2. sterilize the loop 3. sizzle solid media then touch colony with loop 4. mix bacteria into drop of crystal violet 5. resterilize loop and set down 6. use a second slide with no circles and drag it back till it hits the drop and push forward to make a smear 7. throw other slide away 8. let dry 9. decolorize with cooper sulfate 10. dont heat because will degrade capsule 11. blot with bibulous paper * cells stained purple and background with purple tint and blue/no stain on capsule 12. view oil immersion 100 x, turn up brightness, **capsule has no charge so stain doesnt stick to it

total magnification

• Total magnification = Ocular lens Mag (10x) X Objective Mag. • 40X is with the 4X Objective. • 100X is with the 10X Objective. • 400X is with the 40X Objective. • 1000X is with the 100X Objective. • You want enough light to give you a white background. * For Bacteriology, I recommend using the 10X and 100X objective lenses.

Lancefield Classification

•In humans, the majority of Streptococcal infections are caused by β−hemolytic Streptococcus spp. • Carbohydrate antigens found in the cell walls of nearly all β−hemolytic Streptococcus spp. can be used to classify pathogenic Streptococci into groups known as Lancefield Groups. • These antigens are called Streptococcal group specific antigens. • Enzymes can be used to extract these group specific antigens from the bacteria.

Gram Stain

• The Gram stain is the most commonly used differential stain. • It is used to distinguish Gram-positive (will stain purple) from Gram-negative (will stain red/pink) bacteria. ** due to thickness of peptidoglycan layer ( neg thin and positive thick) • The shape and spatial arrangement of the cells is as important as the color of the cells for interpreting this stain. - If you made your smear from a colony on solid medium, the spatial arrangement will not be reliable. Separates bacteria into two classifications according to the composition of their cell walls. Identifies the morphology of the stained bacteria and aids in the interpretation of culture results. Limitations of the Gram stain are: 1) The number of microorganisms required is relatively high, 2) Microorganisms that lack cell walls, such as mycoplasma, cannot be identified using the Gram stain. 1. broth ( barely visible) solid ( hazy) 2. heat fixed smear 3. crystal violet, gram's iodide, acetone alcohol and saffranin 4. flood entire slide with crystal violet ( primary stain) give 1 min 5. rinse or dump off crystal violet 6. gram's iodide ( mortan and binds crystal violet in peptidoglycan layer) flood slide and leave for 2 min 7. rinse or. dump off with forceps or fingers 8. decolorize with acetone alcohol * stronger than acid alcohol ( washes crystal violent and gram's iodide complex from peptidoglycan , quick for negative and. longer for gram positive; if to. long then decolorize all. cells). for 4-6 or 5 seconds. hold slide at angle over tray with wash bottle with deionized water ready **critical timing step 9. rinse throughly with deionized water 10. set slide on try and flood slide with safranin for 1 min ( counter stain); negative with hold to safranin; any cells decolorized will be stained with safranin and will go in positive but crystal violet remains in and pink from safranin wont show 10. rinse at angle into slide tray 10. blot dry with bibulous paper book and examine under oil immersion; negative is red/pink and positive is blue/purple; wipe down microscope, grab by handles, plug in microscope, turn on, push stage high, push brightness to 10x, clean lenses with lense paper and cleaner, move eyepieces to see one circle, turn up brightness, 10x , kohler illumination, focus on white circle, close field diaphragm, polygon, adjust height of condenser so edges clear, center polygon with condenser centering knobs, reopen field diaphragm till edges just. out fo field of view, then do 400x total mag, inc brightness with slider, 10 x lense, oil immersion, 100x ( dont pass through 40x), focus with fine focus ** look near end of smear cocci-circle -bacilli-rod in lab- gram neg bacillius and positive cocci ** modeled staining some pink. areas but mostly purple because poorly constructed peptidoglycan layer ( gram positive cells bacillus. serius; rod shaped) - got area where cells are spread out and not piled on top of each other pull slide off, put into slide box, lower stage, clean. microscope with lense paper and cleaner, biohazard bag, lower light, 10x, unplug and turn off, clean eyepiece, disinfect, plug in holder, cover micrscope, back in cabinet with handles eyepieces first

Catalase Positive vs. Catalase Negative Gram Positive Cocci

When you have a Gram Positive Coccus that is Catalase Positive, you should start thinking: Ooh, a Staphylococcus spp. Maybe a Micrococcus spp. The special tests for Gram Pos. Catalase Pos. Cocci will include tests designed to identify Staphylococcus spp. Mannitol Salt Agar (previously discussed). Coagulase Test Novobiocin Susceptibility Test When you have a Gram Positive Coccus that is Catalase Negative, you should start thinking: Ooh, a Streptococcus spp. Or, maybe it's an Enterococcus spp. The special tests for Gram Pos. Catalase Neg. Cocci will include tests designed to identify Streptococcus spp. Bile Esculin Agar (previously discussed) . CAMP Test Bacitracin Susceptibility Test Optochin (ˈɒptətʃɪn, or op-tah-chin) Susceptibility Test

Selective medium

'selects' for certain organisms, or types of organisms. A Selective medium contains one, or more, components that specifically inhibit the growth of certain organisms, or types of organisms, while allowing the growth of other organisms. A Selective medium contains one, or more, inhibitors. Example: 7.5% NaCl Agar; halophile and nonhalophile

In VICI 325, what lens requires oil? Why do we need to use oil to view microbes?

100X lense requires oil immersion in order to enhance resolution so the very small bacteria or microorganisms can be seen clearly. Don't forget it decreases the amount of diffraction or bending of light rays because it has the same refractive index as glass Some 100x objective lenses are oil immersion lenses (Figure 2a). A drop of oil is placed on the microscope slide and the oil immersion lens is rotated into position. The oil fills the gap between the specimen and the lens and decreases the amount of diffraction or bending of light rays because it has the same refractive index as glass

bacteria smear example

10x or 4x before moving slide on/off so you dont scrape side - use 10x; look like dust as slide (yellow) - focus with fine or coarse - move stage around with mechanical stage control knobs - move to 40x & there is less light that enters so add more light with brightness knob or slide iris diagphram to left ( white background) - use 100x when there is oil immersion objectives so we will add a drop of oil to slide- dont put back to 40x just got to 10x or else it will get dirty and its not made for oil **dont raise and lower stage when moving to different objectives - adjust brightness and slide to left to increase light and use fine adjustment knob to put it into focus **. use lense paper to clean off oil from microscope and eyepieces - put back into cabinet with handles facing you after plugging in microscope first thing to do is put it in kohler to get best quality images

kohler illumination

10x objective lense 1. close field diaphragm by turning counterclockwise ( ridge on field diaphragm) * keep specimen still in focus 2. focus condenser while looking through. eyepieces with left hand -polygon with distinct edges with condenser focus knob 3. center condenser with condenser centering knobs ( usually move left a little to be centered when reopen field diaphragm) 4. reopen field so polygon just inside field of view and then open until just outside field of view. using field diaphragm so polygon just bigger than circle; levae condenser focus knob alone when in kohler 5. use coarse and fine focus knobs and mechanical stage knobs to move slide 6. use iris diaphragm lever of brightness adjustment knob to change brightness *provide the best possible image quality when looking at microorganisms. • Take your compound microscope out of the cabinet using the built-in handles in the back of the microscope. • You will also need to grab a condenser and field diaphragm from one of the pink or purple boxes in the middle of your bench. Each piece will simply click into place. If you are having difficulties, ask the lab staff for assistance. Set up the Microscope and turn on the light source . • Place one of the provided slides on the microscope stage. • Open the Iris Diaphragm Control completely, and set the l0X objective (yellow ring) • Using both ocular lenses, focus on the tissue slide provided (white slide box). • Don't touch the focusing knobs for the rest of this procedure. Focus the Light on the Slide • Close the Field Diaphragm completely (turn counterclockwise); you should see a small circle (octagon) of light. If you do not see the small circle, ask the lab staff for assistance. Focus the Condenser so that edges of the circle are sharp. • Use the Condenser Centering Knobs, center the circle of light in the field of view (if needed) • Open the Field Diaphragm so that the light just fills the field of view. • Re-center the Condenser, if necessary.

Culture Media Forms

A bacteriological culture medium is a mixture of nutrients for growing bacteria. Liquid Medium: • A broth (contains no agar). - Used for maintaining pure cultures and physiological studies . Solid Medium: -Broth mixed with agar to solidify the medium . Agar slant tubes - Used for maintaining pure cultures and physiological studies -take broth add agar, cools, sterilize, solidify and tip tube when cooling . Agar deep tubes - Used for physiological studies - keep tube upright when cooling . Agar plates - Used for isolating pure cultures, maintaining pure cultures and physiological studies.

Nutrients: Chemical & Energy Requirements

All cells require three things to conduct metabolism: - A carbon source - A source of energy - A source of electrons or hydrogen atoms.

oxidase alternative procedure

Alternative Procedures: If you have your bacteria growing on a basic nutrient medium without dyes or pH indicators, you can add oxidase reagent directly to a colony on the plate. • If you want to use the colonies on the plate for inoculating other tests, do not add oxidase reagent to the plate. Some people like to use a cotton swab to pick up a colony, and then add a drop of oxidase reagent to the sample on the swab. • This makes comparison to positive and negative controls more difficult, but some people like to run the test on a swab Does the organism use the enzyme cytochrome oxidase c as the final enzyme in the electron transport system. When you have a Gram - bacillus, do an oxidase test. If the organism is oxidase - it is most likely a member of family Enterobacteriaceae.

Nitrate Test pt.2

After incubation we add 5 or 6 drops (manual says 3 or 4) of Nitrate Reagent A and 5 or 6 drops of Nitrate Reagent B. Give the reagents 2 or 3 minutes to react. • If the broth begins to turn pink (eventually red), there is Nitrite (NO2 - ) present. -The organism is Nitrate (NO3 - ) Reductase positive. -It reduces Nitrate (NO3 - ) to Nitrite (NO2 - ) and Nitrite (NO2 - ) has accumulated in the medium. • If there is no color change after 2 or 3 minutes, there is no Nitrite (NO2 - ) present. -You need to determine if the organism is Nitrate (NO3 - ) Reductase negative [Nitrate (NO3 - ) is still present], or if the organism is Nitrate (NO3 - ) positive and Nitrite (NO2 - ) Reductase positive [there is no Nitrate (NO3 - ) or Nitrite (NO2 - ) present in the medium] Zinc (Zn)can act as a catalyst and will reduce Nitrate (NO3 - ) to Nitrite (NO2 - ). If there is no color change after the addition of Nitrate Reagents A & B, we add a small amount of Zn dust. • Use a 6" applicator stick to transfer a small amount of Zn dust from the little stock tube to your Nitrate Broth. • If you attempt to just dump Zn dust into your broth, you will use an excessive amount, waste my Zn dust, turn your broth dark gray, and irritate me because you ignored my instructions. 3 Possible results: Tube 1: After incubation, Nitrate Reagents A & B were added. The broth turned red. The organism is Nitrate (NO3 - ) Reductase positive. Tube 2: After incubation, Nitrate Reagents A & B were added. The broth did not change color, which means there was no Nitrite (NO2 - ) present. After Zn was added, the broth turned red. Zn reduced the Nitrate (NO3 - ) to Nitrite (NO2 - ). The organism is Nitrate (NO3 - ) Reductase negative. Tube 3: After incubation, Nitrate Reagents A & B were added. The broth did not change color, which means there was no Nitrite (NO2 - ) present. After Zn was added, the broth did not change color (no red). There was no Nitrate (NO3 - ) present either. The Zn would have reduced it to Nitrite (NO2 - ), which would have reacted with the reagents and the tube would have turned red. The organism is Nitrate (NO3 - ) Reductase positive and Nitrite (NO2 - ) Reductase positive. This is a Denitrifying organism. The blue circle is intended to highlight a bubble of gas in the Durham Tube. That would be Nitrogen gas (N2).

Once you have focused on your specimen using the 10X objective, the next step in setting up Kohler illumination is:

Close the field diaphragm by turning it counter clockwise.

Complete, or Beta (β) Hemolysis

Complete, or Beta (β) hemolysis, involves the lysis of rbc's and complete degradation of the hemoglobin from the rbc's. The red color is removed from the media . There will be a circle under each colony where the medium is clear and straw colored. zone where red has been removed - relatively straight through the medium

Bacterial Growth Curve

Lag phase - • Bacteria introduced to a new environment (as when culture medium is inoculated) . • Cells absorb nutrients, adjust to the new conditions (pH, temperature, osmolarity). Log (or Exponential) phase - • Nutrients are effectively unlimited in availability. • Cells are dividing at the maximal rate for the organism. Stationary phase - • Nutrients are becoming limited. • Waste products accumulating in the environment are making the environment less favorable. • The rate of new cell production is balanced by cell deaths. Death (or Logarithmic decline) phase - • Environmental conditions are unfavorable for growth. • Nutrients are present in very limited quantities or no longer available in the environment. • The rate of new cell production is lower than the rate of cell death.

Growth Requirements

Nutrients: Chemical and Energy Requirements - Sources of Carbon, Energy, and Electrons - Oxygen Requirements - Nitrogen Requirements - Other Chemical Requirements Physical Requirements - Temperature - pH - Physical Effects of Water

basic stain

Procedure 1. Prepare heat-fixed smears from a pure culture. 2. Heat-fix the slide. 3. Put 2-3 drops of a stain on the smear and let it sit for 2 minutes. Use crystal violet (1 minute) or methylene blue (1 minute) or safranin (1-2 minutes) . 4. Rinse gently with tap or distilled water. 5. Blot between sheets of bibulous paper. (CAUTION): Too much pressure will break the slide; a "wiping" motion will remove the smear.) 6. Place on microscope and observe using all powers of your microscope, including oil immersion. (If adequately blotted, it is not necessary to use a cover slip, even on oil immersion.)

dentification of Gram Negative Bacilli

The IMViC battery of tests is primarily used for identification of organisms suspected to be members of the family Enterobacteriaceae, but can also be useful for identification of other Gram Negative bacilli. IMViC: Indole production test (Indole Test) Methyl Red Test Voges-Proskauer Test Citrate Utilization Test (Citrate Test)

No Hemolysis

The majority of organisms we will talk about are nonhemolytic. The bacteria may use nutrients from the blood in the medium, but they ignore the rbc's. Some people refer to this as Gamma reaction. gamma-hemolysis- not a good term -no hemolysis then it doesn't make sense to call it that - gamma hemolysis- equals. no hemolysis colonies paired are similar in size and not as. big because of competition and if we pick off of medium there would be no difference in. red color under and. around colonies

Cleaning the Oil Immersion Lens (100x Objective)

The oil immersion lens does not have to be cleaned until the End of the Laboratory Session! When you are ready to put away your microscope, you must clean the 100x objective as follows: 1. Turn off the Microscope!!! 2. Use Lens Paper to wipe off any excess oil from the 100x objective and slides. 3. Dip a cotton swab in the cleaning solution provided. Warning! The Cleaning Solution Is Highly Flammable! Do Not Breathe in the Fumes!!! 4. Use the end of the cotton swab to clean the 100x objective lens, removing any excess oil.

ASEPTIC TECHNIQUE & TRANSFER OF MICROORGANISMS

This involves: Removing an Inoculum from a broth or an agar culture; Transferring the inoculum into broth tube; Transferring the inoculum into an agar plate and streaking for isolation (Figure4)

For the microscopes in VICI 325, why do we perform 'Kohler Illumination' ? What does this achieve?

Your Answer: Kohler Illumination provides the best possible image quality when looking at microorganisms.

Enriched growth medium

is a growth medium supplemented with additional nutrients to support the growth of a wider variety of microorganisms. Fastidious organisms are organisms that require an enriched medium to grow. They are, effectively, little prima donnas that are not capable of synthesizing certain nutrients that they require for growth. Non-fastidious organisms can synthesize what they need from basic nutrients (salts, carbohydrates, lipids, proteins). Streptococcus spp. tend to be fastidious. Escherichia coli is non-fastidious, it doesn't care what you feed it, it'll grow on almost any growth medium. Example: Blood Agar

Microbial Growth

• Microbiologists use the term growth to indicate an increase in a population of microbes rather than an increase in size. • Microbial growth depends upon the metabolism of nutrients, and results in the formation of a discrete colony, an aggregation of cells arising from a single parent cell, or a biofilm, a collection of microbes living in a complex community on a surface

Inoculation Techniques

• We have inoculating needles (a) and inoculating loops (b). • The difference is that the inoculating loop has a ring at the end of the wire. - The loop will allow you to transfer a drop of broth culture. The needle could be used, but you won't get as much bacteria when your source culture is a broth . • In general, we use an inoculating needle when we want to stab the organism into solid medium. In that case, we usually want to put the organism into an anaerobic environment and the loop would leave a gaping hole. • When inoculating bacteria into a broth or onto a solid surface, either the loop or the needle will work. The loop will pick up and transfer a larger inoculum and is less likely to tear the surface of solid media. • If you are stabbing solid medium, use an inoculating needle. • If you want to put bacteria onto a solid surface or into a broth, use either the loop or the needle (hint: I prefer the loop unless I have to use the needle).

Hemolysis

- lysing blood cells for fun and profit Some organisms produce hemolysins that will lyse red blood cells (rbc's). Most hemolysins are phospholipase enzymes which break down the phospholipids of rbc cytoplasmic membranes. - phiodpjage heads on outside. surface round membrane. ( phospholipid membrane) Some hemolysins are porins which aggregate and create channels through the rbc cytoplasmic membrane, allowing the cell's contents to leak out. Most organisms that break open rbc's will also enzymatically degrade the hemoglobin that is released. The goal is get nutrients from the rbc's, especially the iron from the hemoglobin molecule most are enzymes, break rbcs agar addition to enrich agar and nonfastiduous grow better too, better. result for fastidious organisms org use components of plasma and rbcs to use in. metabolism, break up Hb and use iron atom at center

To use the oil immersion objective

1. Focus your specimen using the 40x objective and align the microscope for Köhler illumination. 2. Rotate the revolving turret to the 4x objective or to a position BETWEEN the 40x objective and the 100x oil immersion lens. 3. Place a small drop of immersion oil over the cover glass in the circle of light that can be seen passing through the specimen. Ensure that there are no air bubbles in the oil. Air bubbles will produce a poor image. To get rid of air bubbles, add more oil, or rotate the 100x objective through the oil droplet several times. 4. Rotate the revolving turret so that the 100x objective is in place. Focus the specimen. 5. When you are done, Ensure That You Do Not Rotate the 40x Objective through the Oil!!! If Any of the Other Objectives Contact Oil, Inform the Instructor Immediately! They will clean the objective, or instruct you on how to clean it.

Staining Methods

1. Simple staining: Methylene Blue in Corynebacterium diphtheriae 2. Differential Stain: Gram stain 3. Acid-Fast stains: Ziehl-Neelsen stain; Kinyoun stain 4. Fluorescent Stains: Rhodamine-auramine; Acridine orange 5. Fungal Stains: Lactophenol cotton blue, Periodic acid-Schiff (PAS) 6. Antibody-conjugated stains: Fluorescein isothionate

You can use the 40X objective lens (400X total magnification) to make useful observations of bacteria.

False

Simple Stain

You can choose whatever stain you like: Crystal Violet, Safranin ( red/pink) , Malachite Green, Carbol-Fuchsin, Methylene Blue. -bacillus- long threadlike; rod All cells will be stained the same color, whatever color the stain is. With a simple stain you can determine the size and shape of the cells and perhaps the arrangement of cells the organism prefers, but that is about all you can determine. Put the air-dried, heat-fixed slide on your staining tray. Flood the slide with stain. Let it sit for at least 1 min. Rinse with deionized water. Blot dry with bibulous paper by putting inside book of paper. Do not rub. Rubbing will take your cells off the slide. Put the slide under the microscope and enjoy. **can see solid but not broth

Carbohydrate Metabolism

Fermentation is characterized by substrate phosphorylation - Adenosine triphosphate (ATP) formation is not coupled to electron transfer - An organic is used as the final electron acceptor (e.g., pyruvate) in the Electron Transport Chain . - Specific metabolic end products are synthesized, which may aid in the identification of bacterial species. *carb breakdown and form or organic cmolecules Respiration refers to the method of obtaining metabolic energy that involves oxidative phosphorylation - ATP is formed during electron transfer and the associated reduction of gaseous oxygen in aerobic respiration. - A cell membrane Electron Transport Chain composed of cytochrome enzymes, lipid cofactors, and coupling factors is used during this process. - An inorganic atom or molecule (O2, possibly NO2 - , S, PO3 - ) *O2 final e acceptor *inorganic final e acceptor ( O2 or nitrate, phosohate;ion. or molecule look like O2 instead of O2)

Glucose Catabolism vs. Electron Acceptors

Final hydrogen acceptor = Final electron acceptor glucose-> carb met * bact use gluc-> pyruv ( glyc) ferment- pyruv-> organic e acceptor -> 2 ATP and organic byproducts ( 4ATP per gluc) resp- pyruv-> CAC-> ETC-> O2 final e acceptor (38 ATP per gluc) --some use other inorganic (anaerobic respiration) than O2 but get less ATP **not very important

Dark-field Microscopy Fluorescent Microscopy

In dark-field microscopy the specimen appears luminous against a background of little or no light. Light from below the specimen is blocked so that only light from the outer edges reaches the object at a sharp angle. The object reflects and scatters light near its edges. The scattered light is then viewed through the objective. The dark-field is used primarily to view spirochetes, which do not stain well and are too narrow to be observed by any other methods. Typical organisms include Treponema sp., Leptospira sp., and Borrelia sp. (Figure 9). Fluorescent microscopy uses dyes known as fluorochrome that absorb light in the ultraviolet range, fluoresce, and then emit visible light of a greater wavelength. The fluorescing object appears bright against a dark background. Illumination is provided by high intensity mercury arc or xenon lamps. The light passes through excitation filters that select and limit the wavelength of the transmitted light. Only those wavelengths that can excite the fluorochrome being used are selected. The dichromatic splitter then transmits excited wavelengths to the objective. If the fluorochrome dye is bound to the specimen, fluorescence results. The longer wavelengths that are emitted pass through the beam splitter and the image fluoresces. When the fluorochrome dye is not bound, no fluorescence occurs, and the object remains dark.

CULTURE MEDIA

It is possible to maintain bacteria in the laboratory by using suitable culture media. A culture medium is a solid or liquid preparation used to grow, transport, and store microorganisms. To be effective, the medium must contain all the nutrients the microorganism requires for growth. Culture media can be constructed completely from chemically defined components (defined media or synthetic media) or may contain constituents like peptones and yeast extract whose precise composition is unknown (complex media). Liquid culture media, also known as broth, can be solidified by the addition of agar, a complex polysaccharide from red algae.

Methyl Red Test/Voges-Proskauer (VP) Test

Methyl Red Test - To test the ability of an organism to produce and maintain stable acid end products from glucose fermentation and to overcome the buffering capacity of the system. procedure- 1. sterilize loop; inoculate 2 vials Voges-Proskauer (VP) Test - To determine the ability of some organisms to produce the intermediate product, Acetoin (acetylmethylcarbinol), during glucose fermentation procedure- 1.sterilize loop; inoculate 2 vials **inoculate 2 tubes per organisms and then ass methyl red and VP reagents in one tube

Temperature

Microbes are described in terms of their temperature requirements as (from coldest to warmest): • Psychrophiles grow best at temperatures below 20 °C. (fish pathogens) • Mesophiles grow best at temperatures ranging between about 20 °C and 40 °C. (pathogens because around body temp) • Thermophiles grow best at temperatures above 45 °C. • Hyperthermophiles require temperatures above 60 °C.

Physical Effects of Water

Microorganisms require water (hydrolysis) to dissolve enzymes and nutrients and to act as a reactant in many metabolic reactions. Osmotic pressure restricts cells to certain environments. • Obligate halophiles require high osmotic pressure such as exists in the Great Salt Lake/ high NaCl . • Facultative halophiles do not require but can tolerate salty conditions . • Organisms that live under extreme pressure are called barophiles; ocean floor

ISOLATION OF PURE CULTURES

Pure cultures usually are obtained by isolating individual cells with any of three plating techniques : - The spread plate - The pour plate methods - The streak plate method The streak plate method is routinely used in the laboratory and it is carried out as follows : a. Lift the edge of the lid just enough to insert the loop. b. Streak the loop across the surface of the agar medium using the pattern shown In order to avoid digging into the agar as you streak the loop over the top of the agar you must keep the loop parallel to the agar surface. Always start streaking at the "12:00 position" of the plate and streak side-toside as you pull the loop toward you (Figure 8). As you follow the streaking pattern, each time you flame and cool the loop between sectors, rotate the plate counterclockwise so you are always working in the "12:00 position" of the plate. This keeps the inoculating loop parallel with the agar surface and helps prevent the loop from digging into the agar.

ODC (Ornithine Decarboxylase)

Moeller's Decarboxylase Broth You can add any amino acid you want to test. The presence/absence of Ornithine Decarboxylase is useful in identifying bacteria in the family Enterobacteriaceae. Substrate: Ornithine pH Indicator: Bromocresol Purple Mineral oil covering the surface of the broth preserves anaerobic conditions. Decarboxylase enzymes are activated by acidic, anaerobic conditions. The broth contains glucose as a food source for the bacteria. To grow under anaerobic conditions the organism must be saccharolytic. • Ornithine Decarboxylase Positive (+) - Turbid purple color indicates an increase in pH. - Alkaline (K). - Putrescine is produced when Ornithine is decarboxylated. - Occasionally, E. coli may reduce the indicator to a light gray color; if this occurs you could add a drop of bromocresol purple directly to the tube and reinterpret. • Ornithine Decarboxylase Negative (-) - Bright clear yellow color indicates a decrease in pH. - Acid (A). - Glucose has been fermented with no other activity Ornithine Decarboxylase Positive (+) - You have a saccharolytic facultative anaerobe. - The bacteria ferment the glucose and produce acidic waste products causing a decrease in the pH of the broth. - Ornithine Decarboxylase is activated by acidic anaerobic conditions. - The enzyme will remove the carboxyl group from the ornithine (forming putrescine) - Putrescine is an alkaline compound. - The pH is increased and the medium turns back to purple. - Recorded as K or ODC Pos. - Sometimes Escherichia coli will turn the broth greyish purple. That is still a positive result Ornithine Decarboxylase Negative (-) - You have a saccharolytic facultative anaerobe. - The bacteria ferment the glucose and produce acidic waste products causing a decrease in the pH of the broth. - The broth turns yellow/acidic. - ODC Negative Bacteria will NOT produce ODC, hence the pH will remain ↓ (decreased) and the butt will remain yellow. - Recorded as A or ODC Neg procedure 1. 2 vials, sterilize loop, incoluate, un cap pinky, sterilize, incoluate, sterilize, and sterilize loop 2. repeat for second vial **mineral oil on top make use get down to broth and not in oil; screw cap on tight cause ornithine decarboxylase enzyme works better un anaerobic conditions; others we keep a little loose

Sources of Carbon, Energy, & Electrons

Photoautotrophs use carbon dioxide as a carbon source and light energy from the environment to make their own food. Chemoautotrophs use carbon dioxide as a carbon source but catabolize organic molecules for energy. Photoheterotrophs acquire energy from light and acquire nutrients via catabolism of organic compounds. Chemoheterotrophs use organic compounds for both energy and carbon Chemo/photo ( use light or organic compounds for energy) auto/hetero ( use CO2 or organic compounds for carbon source) *not memorize

Voges-Proskauer (VP) Test

Principle - To determine the ability of an organism to produce the intermediate product, Acetoin (acetylmethylcarbinol), during glucose fermentation. - The end product of the pathway is 2,3-Butanediol. Acetoin does not collect in the medium. - Does the organism use 2,3-Butanediol fermentation when it ferments glucose? Purpose - Primarily, to aid in differentiation between genera, or in species differentiation within a genus, among the Enterobacteriaceae. - We will use the Voges-Proskauer Test for all Gram Negative bacilli. - The Voges-Proskauer test is part of the IMViC battery of tests (Indole, Methyl Red, Voges-Proskauer, Citrate) Biochemistry Involved: - Glucose is metabolized to pyruvic acid, the key intermediate in glycolysis. From pyruvic acid there are many pathways a bacterium may follow; production of 2,3-Butanediol, with Acetoin as an intermediate product, is one pathway for glucose degradation in bacteria. - The addition of VP I and VP II reagents will detect the presence of Acetoin. • Add 4 - 5 drops of VP Reagent I (α-naphthol and KOH). • Add 4 - 5 drops of VP Reagent II (Creatine). • Give the tube a gentle shake VP Positive (+) - If the reagents at the surface of the medium begin to turn pink/red after 10 - 15 minutes, Acetoin is being produced. The organism uses 2,3-Butanediol Fermentation to ferment glucose. You get a stronger reaction (color change) the longer you wait. I actually prefer to wait at least 30 minutes before reading the results. VP Negative (-) - If the reagents at the surface of the medium are yellow or brown, there is no Acetoin being produced. The organism does not use 2,3-Butanediol Fermentation to ferment glucose.

Carbohydrate Fermentation Tests

Principle To determine the ability of an organism to ferment (degrade) a specific carbohydrate incorporated in a basal medium and produce acid (saccharolytic) or acid with visible gas (aerogenic). - use gluc sacchrolytic ( or other carbs) and no gluc sacchrolytic - aerogenic- sacchrolytic and secrete CO2 gas as byproduct when ferment glucose Purpose 1. Fermentation patterns are generally characteristic for specific bacterial groups or species; all Enterobacteriaceae are glucose fermenters. 2. Carbohydrate utilization pattern may aid in species differentiation. phenol red carb broth- nutrient and peptone (part digestive proteins); growth medium and add carbs- named after carb in broth (glucose,sucrose,etc) *The glucose broth tube contains an upside smaller tube (Durham tube) to collect any gases that may be produced. (CO2 if ferments glucose) - heat of autoclave will drive O2 out of durham tube and vaccum will suck medium into tube; dont want bubble in there when we start **some put in all broth but we only put in glucose broth *label sucrose v lactose glucose is more obvious because of durham -open v closed tube -mineral oil prevent O2 from going into tube making an anaerobic environment • The carbohydrate tubes are inoculated with the loop. open- aerobic closed tube- with mineral oil makes it anaerobic 1. closed tube, sterilize, pick. up colony, pull cap off, sterilize, inoculate down into green portion and not in mineral oil, sterilize, cap, sterilize 2. open tube, sterilize, pick up colony, pull cap, sterilize, inoculate, sterilize, cap, sterilize loop **sterilize handle. can needle with broth but prefer use loop **durham only in glucose broth not all carb broths glucose broth 1. label 3 tubes 2. sterilize loop, tap colony, sterilize, inoculate broth, sterilize, cap, sterilize loop 3. dont incoulate to the bottom so you dont grab durham tube 4. repeat for other 2 lactose broth 1. 2 tubes label 2. same procedure 3. repeat sucrose broth 1. label 2 2. same procedure 3. repeat

brightfeild microscopy

The specimen is stained, so its image appears dark against a brighter background. The light is usually provided through a tungsten filament lamp. Compound microscopes have two separate lens systems: an objective (located near the specimen) that magnifies the specimen and an ocular or eyepiece that further magnifies the image presented by the objective (Figure 1). The total magnification is equal to the magnification of the objective multiplied by that of the ocular. The function of the condenser is to gather light rays and focus these on the object to be illuminated. The iris diaphragm is an opaque disk with an adjustable aperture that by adjusting it one may improve on contrast of the image.

Citrate Test

Purpose: To determine if an organism is capable of utilizing citrate as it's sole source of carbon for metabolism and growth. Simmons' Citrate agar is used. • A defined medium. We know exactly what is in the medium. • Citrate is the only source of carbon, and, therefore the only carbohydrate in the medium. • There are no proteins or fats in the medium. • The pH Indicator is Bromothymol Blue. To inoculate: Streak the slant Only (Just get the organism in there. We will be looking for any color change as a positive result.) Principle: Can the organism use Citrate as it's sole source of carbon for energy production and metabolism? • Positive (+) - If the organism can use Citrate as its sole source of carbon for energy and metabolism, the medium turns blue (part of the medium or all of the medium). - You may not see obvious growth on the surface of the agar, but if there is blue, it can grow, which means it can use Citrate as its sole source of carbon for energy and metabolism. • Negative (-) - No growth and no change in color, the medium remains green. - The organism cannot use Citrate as it's sole source of carbon for energy and metabolism. pH changes Citrate is a carbohydrate. If the organism can use citrate as it's sole source of carbon for energy and metabolism, it will make acidic by-products and Carbon Dioxide (CO2). CO2 diffuses out of the cells into the medium, where it reacts with Sodium (Na) and water. This secondary reaction yields Sodium Carbonate (NaCO3) which is alkaline. The pH in the medium increases. So, even though the pH in the medium has increased, we have not broken the rule that whenever a bacterium uses a Carbohydrate it makes acid **use 2 organism for 4 tests because give opposite results.( indole, methyl, VP and citrate) procedure- 1. inoculation with loop technique 2. repeat

Aseptic Technique

Putting the bacteria you want, where you want them, without adding bacteria you don't want. Inoculating a broth from a broth: **label tube with sharpie • Make sure the bacteria are in suspension. • Sterilize your loop in the bacti-incinerator - It only takes a few seconds . - The end of the handle where the wire enters the handle should be inserted into to opening of the bacti-incinerator. • Pick up the culture tube with your organism . • Pull the cap with the pinky finger of the hand holding the loop. - Hold the cap in your pinky, don't set down, don't turn it over, don't pass it from one hand to the other. • Pass the top of the tube in front of the incinerator opening 1 or 2 times. • Insert the loop into the medium and gently swish it around a couple times. If it sizzles, swish it around a couple more times. • Pull the loop out of the tube with a drop of the broth in the loop. Inoculating a broth from a broth : • Pass the top of the tube in front of the incinerator opening 1 or 2 times. • Recap the tube . • Pick up the tube of broth you wish to inoculate. • Pull the cap with the pinky finger of the hand holding the loop. - Hold the cap in your pinky, don't set down, don't turn it over, don't pass it from one hand to the other. • Pass the top of the tube in front of the incinerator opening 1 or 2 times. • Insert the loop into the medium and gently swish it around a couple times. (Twirl it if that makes you happy. Just don't spill the medium.) • Pass the top of the tube in front of the incinerator opening 1 or 2 times. • Recap the tube. • Re-sterilize the loop before you do anything else. • Put the loop down in a safe place, or move onto inoculating something else • The loop is hot when you finish inoculating something . • Don't just lay it on the counter. • The base of the incinerator has a series of holes that will allow you to put the smaller handled loops/needles there. • You can stand the loop/needle in a culture tube rack (handle end first). • You can lean the loop on a steel staining tray or on your aluminum culture basket. inoculating slant- place droop of broth on botttom of slant -butt of tube ( anaerobic) and slant is (aerobic) - when you stab use needle - stub bottom of tube in slant tube and coming out wiggle needle on top of slant as you come out -resterilze and cap, resterilze needle * bigger tubes usually stab and streak on top of slant to see difference in growth in the anaerobic and aerobic part of slant tube

Urea Test

Some bacteria have the ability to produce the enzyme Urease. This enzyme hydrolyzes urea leading to the formation of 2 ammonia molecules, one CO2 molecule, and one water molecule. The Urease Test is commonly used to differentiate among bacilli (both Gram + and Gram -;diff cocci too). The lab manual highlights its use for differentiating Proteus sp. from other members of the family Enterobacteriaceae. (NH2)2CO + 2H2O-urease-> CO2+ H2O+2NH3 The Urease test can be performed with either Urea Broth or Urea Agar. We use Urea Broth because it works better in our laboratory. Urea Broth is a growth medium that contains: 1.Glucose - a food source for the bacteria. Many of the organisms we might test are saccharolytic. 2. Urea - the substrate. 3. Phenol Red - a pH indicator. Ammonia forms Ammonium Hydroxide when dissolve in water, making the broth alkaline. Urea Broth is relatively neutral to start (7.4) pH chnage turnsinto color change. To inoculate the medium, use a loop and transfer the organisms to the broth. Incubate for 24 to 48 hours at 37C. The majority of the bacteria we might put in Urea Broth are saccharolytic. They will use the glucose and produce acidic by-products. If they do not produce urease, the medium will become more acidic (A) and turn brighter yellow. If the organism is asaccharolytic and urease negative it will use peptones for food and the medium will become slightly more alkaline due to alkaline by- products and become, shall we say, salmon colored (vaguely pink). If the organism produces urease the medium will become highly alkaline (K) and turn hot pink (both saccharolytic and asaccharolytic bacteria) procedure 1. label 2 2. loop inoculation 3. repeat **label dont turn tube sideways because will spill so turn head sideways urease- ammonia produ-> ammonia hyd alk meadium -hot pink= alkaline; urease + - yellow = ur - and sacch -salmon pink - ur and assach

The Streak Plate Method

Streak for isolation LABEL not lid/cap ** successive streaks to have isolated colonies - pure culture - different colony types and subculture with each colony type we find on the plant - four quadrant technique - label plate on bottom not on lid because lids can get switched - initials, type of culture and date and keep near edge of plate so you can see colonies through plate - agitate broth culture but not up and down - can divide into quadrants and number Broth * don't always have to resterilize in between because broth spread out but recommends it ** only pick up from source culture once and then streak with colonies on streak plate 1. sterilize loop 2. up cap tube with. pinky, sterilize tube, put loop in, resterilize, cap 3. streak bacteria back and fourth in. first quadrant 4. sterilize loop 5. dont go back to original culture, tap primary colony, streak second quadrant, resterilize 6. touch secondary streak and speared into third quadrant, sterilize 7. repeat with quadrant four, sterilize and store solid 1. label plate with sharpie, date 2. divide plate into quadrant with sharpie 3. sterilize loop, touch colony on solid media, streak quadrant one, resterilize 4. touch quadrant one colony, streak quadrant two, resterilize 5. repeat for quadrants three and four ( fourth goes towards center of plate and not just on edge like previous quadrants) ** more important to resterilize when using solid rather than broth each streak decreases amount of cells -each colony from a colony forming unit - form each colony from a single bacterial cell-> clone-> colony -some cells stay in groups and colonies can from a couple of cells rather than single cell ex; primary= confluent/ continuous growth 2= few colonies and almost confluent 3= same trend 4= isolated colonies keep plates with lid on table to dec condensation and contamination if colonies dont look the same then think its a mixed culture rather than a pure culture - try to subculture to isolate each different colonies to try and get a pure culture to identify colony not a long a streak so a contaminant from the atmosphere

structure

eyepiece/ocular lens- look through ( 10 x mag) head of microscope knob. for adjusting microscope head- on right side to tighten/loosen head of microscope power switch power cord storage brightness adjustment- more/less power to light; keep at middle turret with objective lenses- switch objectives by turning turret; dont push lense itself red- 4x yellow- 10x blue- 40x black- 100x stage- where slides sit mechanical stage control knobs- move stage stage clip- clips slide to stage and moved by mechanical stage control knobs condenser- cone shaped lense and in middle is glass field diaphragm- ridge on ring to inc or dec diameter of light coming out of light source iris diaphragm control - inc dec light coming from condenser - numbers; low power turn to right and higher mag move towards left to let more light to come through smaller area condenser centering knobs- 45 degree angles to move condenser around coarse and fine focus knobs- on left side, coarse bigger than fine - use left hand to focus and move stage up and. down and use right hand to move slide around condenser focus knob- under left side of stage, turn to move condenser up and down independent of stage.

Eosin Methylene Blue (EMB) Agar

• Selective/Differential medium used for identifying/differentiating among members of the family Enterobacteriaceae. • Selective: • Eosin and Methylene Blue inhibit the growth of Gram pos. bacteria and most non-enteric Gram neg. bacteria (family Pasteurellaceae). • Differential: • Lactose - Potential substrate • Eosin and Methylene Blue - Act as indicators (in addition to being inhibitory chemicals) - organisms that can grow • Lactose-fermenter - Most lactose fermenters will form blue-black or pink colonies with dark centers. - Fast lactose fermenters (primarily Escherichia coli) will produce colonies with a metallic green sheen nearly every time. • Non-lactose-fermenter - Bacteria will appear colorless or translucent. • Escherichia coli will appear as dark purple colonies with green metallic sheen. • Rarely will you not see at least a portion of the growth of E. coli with a green metallic sheen. • Some strains of other spp. can produce a green metallic sheen . • Until proven otherwise, an organism that produces growth with a green metallic sheen on EMB agar is presumed to be E. coli. It is at least a fecal coliform.

Enriched, Selective, and Differential Media

We use a large number of different media to grow, characterize, and identify microorganisms. As we go through the process of learning about the various media we will use in this course you should concentrate on understanding what is important about each medium. What is in the medium? What can the medium tell us about the organisms that grow, or in some cases don't grow, on/in the medium? Why might we choose a certain medium to inoculate? You don't need to memorize what each and every organism will do on each medium. We will expect you to know the types of organisms that are expected to give certain results on certain media. There will be a small number of tests/media where you will need to know what a certain species of organism will do. After we discuss a variety of media and other tests, we will ask you to use appropriate media/tests to identify an unknown organism. Within reasonable (to us) limits, we hope to simulate what you would do in a diagnostic laboratory when you receive a sample from a real world source. The unknown identification exercises will be a challenge, but you will be ready for it when we get there. Trust me.

MR/VP Medium

• A broth containing 0.5% glucose. • It is used for both the Methyl Red and Voges-Proskauer Tests . • It DOES NOT contain any pH indicator. • It is inoculated and let incubate for at least 48 hours. • Use separate tubes for the MR and the VP tests. That means inoculate 2 tubes of MR/VP Medium. • Inoculate with your loop • Incubate 24 to 48 hours at 37C. The plan is to determine if the organism uses Mixed Acid Fermentation, 2,3-Butanediol Fermentation, or neither, or in rare cases, has the ability to use both pathways After incubation: Let's add some reagents to the VP Tests First: You should have 2 tubes of MR/VP Broth for each organism. • If you don't, you messed up. • If you admit your mistake now, we can fix it. • Say something and we will bring some clean tubes and transfer pipets. -Add 4 - 5 drops of VP-I, and 4 - 5 drops of VP-II to each Voges-Proskauer Test tube. • That means one tube of Escherichia coli in MR/VP Broth and one tube of Enterobacter cloacae in MR/VP Broth. • Put them aside and let them sit. • We will talk to them later. • Because Acetoin is an intermediate product in the 2,3-Butanediol Fermentation pathway, it does not accumulate in the broth. Therefore, we need to give the organism time to make some Acetoin that will react with the reagents we added to the tube.

Selective/Differential medium '

'selects' for certain organisms, or types of organisms and allows us to differentiate organisms that are capable of growth on (or in) the medium. A Selective/Differential medium contains one, or more, components that specifically inhibit the growth of certain organisms, or types of organisms, while allowing the growth of other organisms and one, or more, potential substrates and one, or more, indicators to allow us to determine if the organism uses the substrate(s) or not. A Selective/Differential medium contains one, or more, inhibitors, and one, or more potential substrates and one, or more, indicators. Examples: MacConkey's Agar, Mannitol Salt Agar

Nitrate Broth

A standard growth medium with Potassium Nitrate (KNO3). The broth has a small, inverted glass tube, called a Durham Tube, submerged in the broth. *no bubbles when start • The Durham Tube is intended to capture N2 gas if the organism is a Denitrifying organism, that is, it reduces Nitrate (NO3 - ) all the way to N2. • Before you inoculate the broth you should take a quick look to be certain there are no bubbles in the Durham Tube. After incubation, any bubbles in the Durham Tube should be N2 produced as a result of complete reduction of Nitrate (NO3 - ) by the organism To inoculate the medium, use a loop. • Be careful not to insert the loop all the way to the bottom of the tube, hook the bottom of the Durham Tube and pull the Durham Tube out. • You just need to get the inoculum into the broth. It will make its way around inside the tube. Incubate at 37C (body temp) for 24 to 48 hours. • 24 hours is standard. We use 48 because we are working in a college/university class schedule. procedure 1. durham tubes in there 2. label 3 3. loop inoculation 4. repeat for 2

Motility Test Medium

Flagella are responsible for the motility of bacteria and can play a role in the production of disease. Bacterial cells may carry a single flagellum, and are thus described as monotrichous. If the single flagellum is at one end of a rodshaped cell it is known as a polar flagellum. If the bacterium carries a single tuft of flagella, it is said to be lophotrichous (lophos - Greek for a crest). When the tuft appears at both ends of the cell, the bacterium is amphitrichous (amphi - Greek for 'at each end'). Bacteria that are covered all over in flagella arxe said to be peritrichous (peri - around). Motility in bacteria may be detected by a direct method by staining the Flagellum (a) which allows direct study of flagellar arrangement, but it is tedious, and requires certain skill. Indirect methods may be used, e.g., (1) The Hanging Drop method allows the observation of any movement of the bacteria in a suspended drop of broth, (2) Motility Test Medium Purpose An indirect indicator of the presence of bacterial flagella. The movement of bacteria is detected by use of a medium with low agar concentration. The medium is inoculated with an inoculating needle, incubated with other tube media, and can be read without the need for a special stain and microscope. Principle The lower agar concentration in the medium allows limited movement of motile bacteria from the area of the stab. Motility will be detectable as diffuse growth radiating from the stab line. A special dye,a tetrazolium salt (TTC) may be added to make the radiating growth more visible as the reddish diffuse area Procedure The stab is performed with an inoculating needle, shown at right. Care must be taken to insert the needle straight in and to pull it straight out. If the needle "slices" the agar horizontally, it may be difficult to tell if the resulting bacterial growth is from motile bacterial, or is just growth along a wide stab line. Procedure The stab is performed with an inoculating needle, shown at right. Care must be taken to insert the needle straight in and to pull it straight out. If the needle "slices" the agar horizontally, it may be difficult to tell if the resulting bacterial growth is from motile bacterial, or is just growth along a wide stab line.

Types of Culture Media

General Purpose (Nutrient) Media -They support the growth of many non-fastidious microorganism, e.g. tryptic soy broth and tryptic soy agar. Enriched/Fortified Media -They support and encourage the growth of fastidious bacteria. They are commonly used to harvest as many different types of microbes as it can, e.g. blood agar. Selective Media -They favor the growth of particular microorganisms, while specifically inhibiting the growth of others. Selective media expedite the rapid isolation and identification of particular microbes by preventing the growth of interfering organisms, e.g. Columbia CNA agar. *Some media are selective by: - virtue of limited nutrients - addition of inhibitors that suppress the growth contaminants - addition of antibiotics that prevent the growth of many contaminants. *Highly selective media permit the use of inocula containing a large number of contaminating organisms, as long as the target bacterium is not also inhibited by the medium. Differential media -They are media that distinguish between different groups of bacteria and even permit tentative identification of microorganism based on their biological characteristics. Differential media contain indicators that distinguish between organisms on the basis of their appearance on the medium. Selective/Differential Media -They are wide spread media used in isolation and identification of bacteria. The media combines the characteristics of both the selective media and the differential media, e.g. MacConkey agar, EMB agar, SS agar.

Methyl Red Test

Principle: - To test the ability of an organism to produce and maintain stable acid end products from glucose fermentation and to overcome the buffering capacity of the system. - This is a quantitative test for acid production (pH determination); some organisms produce more acids than others. Purpose - Primarily used to aid in differentiation between genera or in species differentiation within a genus among the Enterobacteriaceae. - We will use the Methyl Red Test for all Gram Negative bacilli. - The Methyl Red Test is part of the IMViC battery of tests (Indole, Methyl Red, Voges-Proskauer, Citrate). Biochemistry Involved - All members of the family Enterobacteriaceae ferment glucose by definition. The different pathways of fermentation are dependent on the nature of the bacteria involved. - Some members of the family Enterobacteriaceae ferment glucose through the mixed acid pathway. These mixed acid end-products lower the pH to <4.4. - At this pH, the pH Indicator Methyl Red is red. - Within the pH range of 4.4-6.0 is varying shades of orange. - At pH > 6.0 Methyl Red will be yellow. The Test: - Add about 6 drops of Methyl Red and give the tube a gentle shake . - You want some of the broth to mix with the layer of Methyl Red on the surface, but you don 't want to completely mix the 2 . - If the Methyl Red remains red, the pH is below 4.5 and the organism is Methyl Red positive. The organism uses Mixed Acid Fermentation to ferment glucose. - If the Methyl Red turns orange or yellow, the organism is Methyl Red negative. The organism does not use Mixed Acid Fermentation to ferment glucose.

Indole Test

Principle: - To determine the ability of an organism to split indole from tryptophan. Indole is a by- product of no real use for the organism, but we can test for it. Medium: Tryptophan Broth Substrate: Tryptophan No pH indicator No other components of interest. To Inoculate: Use your loop. Incubate for 24 to 48 hours at 37C Biochemistry Involved - Some organisms produce Tryptophanase which allows them to break down Tryptophan. Tryptophan ------------> Pyruvic Acid + Ammonia + Indole - The bacteria utilize the pyruvic acid and, in some cases, ammonia for their metabolic needs; Indole is not used and accumulates in the medium. - The presence of Indole can be detected by the addition of Kovacs' Reagent. - Kovacs' reagent reacts with the Indole, producing a bright red compound in the reagent layer at the surface of the medium. The test: -After incubation, add 4 - 6 drops of Kovac's Reagent to your tube. -Don't mix the reagent into the broth. -If the reagent layer turns red within about 1 minute, Indole is present. The organism produces Tryptophanase and can break down Tryptophan with Indole as a by-product . -If the reagent layer does not turn red within about 1 minute, Indole is not present. The organism does not produce Tryptophanase. Important note: Sometimes an Indole positive test will change from red to green after 20 or 30 minutes. This does not mean the organism magically went from Indole positive to Indole negative. Don't change your answer. If it bothers you, add 5 or 6 more drops of Kovac's Reagent and you will get more red. 1. 2 vials incoluate with loop; same procedure as all others 2. repeat

Hydrogen Sulfide Production

Principle: - To determine whether hydrogen sulfide (H2S) gas has been liberated enzymatically from sulfur-bearing amino acids to produce a visible, black color reaction in the presence of a H2S indicator system Biochemistry Involved: - Peptones, cysteine, cystine, methionine, and thiosulfate are all sources of sulfur for certain heterotrophic bacteria. - The enzymes responsible for this activity are cysteine desulfhydrase and thiosulfate reductase. Media & Indicators: - Many peptone iron-containing media are on the market to detect H2S production among the Enterobacteriaceae. - In most of these media sodium thiosulfate act as the source of sulfur, while the indicators are either ferrous sulfate or ferric citrate salts.

smear prep

Procedure (Figure 10) Broth Culture 1. Shake the culture tube and with an inoculating loop, aseptically transfer 1 loopful of bacteria to the center of the slide. 2. Spread this out to about ½ inch area. 3. Allow the slide to air dry, or place it about 12 inches above the flame to dry in hot air. 4. Heat-Fix and kill the bacteria by passing the slide through a Bunsen burner flame three times. Now the slide is ready to stain. Slant or Plate Culture 1. Place a loopful of water in the center of the slide. With the inoculating needle, aseptically pick up a very small amount of culture and mix into the drop of water. 2. Spread this out to about ½ inch area. 3. Allow the slide to air dry, or place it about 12 inches above the flame to dry in hot ai

Incomplete, or Alpha (α) Hemolysis

Some organisms produce hemolsins that will lyse red blood cells (rbc's) with no enzymatic effect on the hemoglobin. The hemoglobin will degrade on its own to methemoglobin, which is green. This results in a circle under each colony that has a green tint to it. The green tint is often very difficult to visualize * break ope cell membrane to get to insides to use for biological process but don't have. enzymes to break down Hb themselves so Hb. releases. into medium and. hemoglobin will degrade by itself when it's exposed to oxygen and light and turn to methemoglobin which is green. **hemoglobin will degrade by itself when it's exposed to oxygen and light and turn the men hemoglobin which is green. So with incomplete or alpha analysis, we have a circle under aids colony that screen, we do have a new our new strain that we got recently. Hb and the diffuse same distance in all. directions, half a sphere with hemolysis -surface- cut. sphere. in. half narrow zone the hemolysis don't diffuse as far out diameter of zone of hemolysis is not correlated to the type of hemolysis The bigger it gets, the straighter the sides. If the diameter tpf zone of heolysis is will say a millimeter across and the medium is four millimeters thick that's going to be a little bit of sphere right near the surface, under the colony, as opposed to if the diameter of that. is will say a millimeter across and the medium is four millimeters thick that's going to be a little bit of sphere right near the surface, under the colony, as opposed to if the diameter of that. So with incomplete or alpha analysis, we have a circle under each colony that green look for isolated colonies. and not primary. streak

Catalase Test

The enzyme Catalase is produced by some bacteria to detoxify peroxide anions (O2 - ) including Hydrogen peroxide (H2O2). Some bacteria use a peroxidase enzyme to accomplish this. In order for an organism to be able to survive in an O2 rich environment (room air conditions) it must be able to detoxify H2O2. These are constitutive enzymes. If the organism can produce one, or both of them, it will do so all of the time so that they will be available for use when needed. Some enzymes are inducible - they are not produced unless/until they are needed A test for the presence of the enzyme Catalase. Primarily used to differentiate between genera. Biochemistry H2O2-> 2H2O + O2 Procedure: Using a clean, sterile toothpick for each organism to be tested, transfer a colony from an agar plate (we use Blood Agar) to a clean glass slide. Add a drop or 2 of 3% H2O2. If the organism produces Catalase, bubbles of O2 will begin to form immediately. If the organism does not produce Catalase, no bubbles will form. When you determine that you have Gram Positive Coccus, the next test to perform is the Catalase Test. Certain tests are more helpful in identification of Catalase Positive Gram Positive Cocci. Certain tests are more helpful in identification of Catalase Negative Gram Positive Cocci . Of the medically important Gram Positive Cocci: The Catalase Positive genera Staphylococcus spp., Micrococcus spp. The Catalase Negative genera Streptococcus spp., Enterococcus spp. Keep in mind that the only Gram Negative Cocci of medical importance are the Neisseria spp. (Human Medicine). There are no Gram Negative Cocci of importance in Veterinary Medicine (no, we don't call it inhuman medicine).

Results- Lab 5 pt.2

add VP reagent (4-5) and set aside -looking for acetonin so give a few minutes - pink to red= positive - no color= negative indole- add Kovac's reagent - color chance instant - red layer on surface= positive indole production from tryptophan - yellow layer= negative indole production **sometimes those who are indole + will go back to yellow after time citrate- - red= grow with cirate as sole source of carbon energy, more K because CO2 produced reacts with salts even though produces salt * if part of tube blue = + -green= no citrate cause bacteria doesnt grow methyl red - mixed acid ferm pathway - add pH indicator methyl red (1-2) - pathway. other than methyl red or assachrolytic turn orange or yellow and pH close to 6 - does use mixed acid ferm pH is below 4.5 and. turns red/pink **no other pathway give that low of ph after incubation glucose ferm- majority have one ferm pathway s o expect either positive M or neg VP or neg M and + VP or neg for both; rarely positive for M and VP lysine iron agar *nothing will do both because wont look like lys anymore- 1. blue slant, yellow butt= K/A - LDA + LDC - - growing sloer in butt of tube still using glucose and on slant ran out of glucose and use peptones turning back to purple 2. K/A + h2S + G 3. K/K - sacc faculative anaerobe - was all yellow but ran out of glucose on slant use peptone and in butt act LDC enz rip CO2 off lys and made cadaverin - LDA - and LDC + **bubbles arent critical for this test 6. K/K + H2S; entire butt is black so cant tell if K or A because masks color * cant determine LDA and LDC result 7. R/A; mid range on slant and acid on butt - was all yellow, ran out of gluc on slant and use peptones prod K products. and gluc use in. butt - LDA ; weak Carb acid- a-keto acid-> weak acid + alkalyne products get a mid range pH change ( port wine) ODC- 1. purple= positive e coli sometimes make gray - sacc ana; yellow -> purple 2. no color change no ODC stays acidic because still using glucose and making acid salmonella agar **must be isolated to tell lactose use 1. isolated colonies clear or yellow-> no lactose growth positive 2. grows and uses lactose- isolated colonies and pink and media pink 3. pos growth and less lactose use so pink colonies but no pink media 4. grows but neg lactose so media is yellow and black colonies - H2S production EMB 1. grows and no lactose= colonies same color as medium 2. grows and uses lactose= pink or blue black colonies ** look at isolated colonies not primary streak 3. shiny green colonies= fast lactose use and fecal maconky- enter baci no other gram - bacilli - growth = gram - - no growth= gram + or some - *isolated colonies - pink colonies or plate is pink= use lactose ( lots of lactose if media is pink too) 2. peptone for food = yellow or colorless and lactose neg

Results Lab 4

glucose broth tube 1- K red/orange (asacc) tube 2- A yellow. (sacc) tube3- A and CO2 prod (sacc and aerogenic) lactose broth tube 1- K tube 2- A and CO2 (aerogenic ) sucrose- tube 1- K tube 2- A TSI tube 1 - sacch fac anaerobe ( anaerobe in butt and aerobe on slant). & ( faster on slant so lots of gluc use and then ran out and use peptones so made it red than butt is yellow cause using gluc) - yellow-> K/A tube 2/3- all yellow A/A (finger trick); E coli aerogenic cracks or bubbles tube 4- obligate aerobe asacch K/N tube 5- H2S black in bottom so acidic if at butt -gas bubbles at bottom so CO2 use carbs; sacc -slant is red and with finger rule dont know bottom of slant is K or A -slant is thinner - dont use lac,suc or gluc then all red- use peptones -compare to other broths then use gluc and not lac or suc -K/A + G + S **K on slant because isnt using on of dissachrides tube 6- black at bottom ** A/A +G + S because used one of diasschrides so A on slant even though cant see bottom of slant tube7- bottom of slant is yellow A/A; black between alkaline and acid; tube 8- weak H2S -A/A bec use suc and gluc urease - tube 1- pink- ur + K reaction -tube 2- pale or bright yellow- ur neg A nitrate * add reagent A and B 4 drops each -tube 1- bubble= denitryfiying bacc prod N2; add Zn no color change so no NO3 or NO2 -tube 2- pos nitrate-> color change - tube. 3- added Zn color change-> nitrate neg no NO2 , NO3 present

MacConkey's Agar

inhibitory chemicals: Bile Salts and Crystal Violet Substrate: Lactose pH indicator: Neutral Red Used in the identification of Gram - bacteria (primarily for members of the family Enterobacteriaceae). Gram + organisms are inhibited by the crystal violet. Some non-enteric Gram - organisms are inhibited by the Bile Salts. Bile Salts tend to be inhibitory to non-enteric organisms regardless of their Gram Staining characteristics (cell wall structure). All members of the family Enterobaceriaceae will grow on this medium. Lactose users will produce pink to red centered colonies. Some also turn the medium around the colonies pink. The pH is decreased as the organism uses lactose and produces acidic by-products. Non-lactose users will produce yellow colonies and turn the medium around the colonies yellow. The pH is increased as the organism uses peptones for food instead of lactose (the only carbohydrate available in this medium).

Eosin Methylene Blue Agar (EMB agar)

inhibitory chemicals: Eosin and Methlyene Blue Substrate: lactose Indicators: Eosin and Methlyene Blue Used primarily to differentiate among members of the family Enterobacteriaceae. All members of the family Enterobaceriaceae will grow on this medium. If you have a Gram - bacillus that is oxidase - , it is almost always a member of the family Enterobacteriaceae. Lactose - organisms produce clear colonies. Most Lactose + organisms produce pink or Blue/black colonies. E. coli produces Shiny green metallic colonies. The growth of shiny green metallic colonies almost always means you have E. coli, but always means the sample contains a fecal coliform bacillus procedure 1. 3 plates 2. streak for isolation with same technique

Differential medium

is a medium that allows us to differentiate between 2 or more organisms. A Differential medium contains one, or more, potential substrates and one, or more, indicators to allow us to determine if the organism uses the substrate(s) or not. Generally, we look for a color change. Examples: Triple Sugar Iron Agar, Phenol Red Glucose Broth, Simmon's Citrate Agar

Microbiological growth medium

is a mixture of nutrients intended to support the growth of one or more microorganisms. A liquid growth medium is called a broth. Essentially, we boil the snot out of some nutrient source (i.e. soy beans), strain it, sterilize and we have a broth. To make a solid medium we add agar agar(an extract from seaweed that we usually just call Agar), or, in some cases, gelatin (collagen from animal tissues). ; some can break down collagen protein so use agar because cant break down Examples: Tryptic Soy Agar, Tryptic Soy Broth, Potato Infusion Agar

Phenol Red Carbohydrate Broth/Carbohydrate Fermentation Tests

pH Indicator: • Phenol Red Substrate: • A carbohydrate - Broth is named for the carbohydrate (i.e. Phenol Red Glucose Broth, Phenol Red Lactose Broth). • Any carbohydrate can be incorporated for testing. Durham Tube: • Glass tube upside down in the broth. • Allows detection of CO2 production from the CHO. • We use a Durham tube in Phenol Red Glucose Broth • Positive carbohydrate reaction: - Acid by-products lead to ↓ in pH, causing the phenol red to change from red/pink → yellow (A). - The inverted Durham tube in the glucose broth is used to collect any gas produced (A+G). • Negative carbohydrate reaction: - The utilization of proteins in the medium leads to ↑ pH, causing the phenol red to change to a darker red color (K). 1. sterilize loop 2. touch mediium, pick up colony 3. pinky cap, sterilize tube, put loop in broth by durham, wiggle 4. sterilize tube, cap, resterilize loop

carb Results Lab 4

phenol red carb broth -phenol red pH indicator - one carb (glucose, sucrose, and lactose) -durham tube allows detection of CO2 (aerogenic)production. from the. CHO ; in glucose broth *positive- dec pH red->yellow (A) *negative- inc. pH red-> darker red (K) TSI- .1 glucose, 1. lactose, 1 sucrose, sodium thiosulfate (sulfur source prod H2S gas) -phenol red pH and ferrous. sulfate indicator for H2S *red/yellow- alkaline/acid - faculative anaerobe; sacchrolytic and uses glucose only -yellow in but. is result of. diff quantities of the sugars -K/A -incubation -> yellow (gluc + fac anaerobe)-> K/A peptone use in slant ( aerobic) and gluc in butt (anaerobic) -run out of gluc and cant use lac or suc then use peptones for food on slant and back to alk because alk byproducts *yellow- A/A -fac anaerobe, sacc and ut glucose and lac and/or suc -bubble= Co2 ; aerogenic -yellow (gluc + fac ana)-> yellow use suc and or lac (not red because can use lac and suc and not peptone use and turn to red) *red/no change- K/N - fail to ferm gluc and aerobic dont grow in. butt (assach because only use peptones on slant; darkening doesnt mean h2S bec bottom hasnt turned balck) and no CO2 prod because dont use gluc *black precipitate- H2S prod under acidic anaerobic cond - H2S react with iron to form iron sulfide->. black precipitate -butt is yellow and acidic ( sacchrolytic fac anaerobe) sulfur req acidic enviornment so some fermentation occurs - bottom has black so look at broth and compare

Lysine Iron Agar (LIA)

rimarily used to identify organisms in the family Enterobacteriaceae (saccharolytic facultative anaerobes). Substrates: Lysine (an amino acid) Sodium Thiosulfate (source of sulfur for H2S production) pH Indicator: Bromocresol Purple H2S Indicator: Ferric ammonium citrate Also contains 0.1% glucose as a food source (leads to acidification of the butt of the tube). It should be noted that this is a limited amount of glucose (similar to TSIA). Slant tube: To inoculate, stab the butt and streak the slant *produce LDA LDC or H2S *gram negative neg catalse entero - purple= K - yellow = A Purple Slant/Yellow Butt = K/A or Alkaline over Acid - Purple Slant • LDA (-) no net change in pH - Yellow Butt • LDC (-) ↓ pH -only glucose LDA Negative (-), LDC Negative (-) You have a saccharolytic facultative anaerobe. - The bacteria use the glucose and produce acidic waste products throughout the medium. The pH ↓ (decreases) and the medium throughout the tube turns yellow. - The organism grows faster on the slant, with O2, and slower ***********, without O2. - When the organism runs out of glucose on the slant, it begins using peptones for food and begins producing alkaline waste products (NH3). The slant turns back to purple. - Because the organism continues to use glucose ***********, the butt will remain yellow. Purple Slant/Purple Butt = K/K or Alkaline over Alkaline - Purple Slant • LDA (-) No net change in pH. - Purple Butt • LDC (+) No net change in pH DA Negative (-), LDC Positive (+) - You have a saccharolytic facultative anaerobe. - The bacteria use the glucose and produce acidic waste products throughout the medium. The pH ↓ (decreases) and the medium throughout the tube turns yellow. - The organism grows faster on the slant, with O2, and slower ***********, without O2. - When the organism runs out of glucose on the slant, it begins using peptones for food and begins producing alkaline waste products (NH3). The slant turns back to purple. - Acidic, anaerobic conditions *********** of the tube activate the enzyme Lysine Decarboxylase, which removes the carboxylic acid groups from the Lysine molecules. - The resulting amino compound is alkaline, and the butt turns back to purple. Portwine (maroon) Slant/Yellow Butt = R/A or Red over Acid - Portwine Slant • LDA (+) ↓ pH - net slight decrease in pH of slant. - Yellow Butt • LDC (-) ↓ pH - decreased pH of butt DA Positive (+), LDC Negative (-) - You have a saccharolytic facultative anaerobe. - The bacteria use the glucose and produce acidic waste products throughout the medium. The pH ↓ (decreases) and the medium throughout the tube turns yellow. - The organism grows faster on the slant, with O2, and slower ***********, without O2. - When the organism runs out of glucose on the slant, it begins using peptones for food and begins producing alkaline waste products (NH3). The slant starts to turn back to purple. - Lysine Deaminase (LDA) removes the amino group from lysine molecules producing a weakly acidic product (an a-keto acid). - The weak acid from the activity of Lysine Deaminase and the alkaline waste products from using peptones for food reduce the pH in the slant slightly, causing the slant to turn Port Wine/Maroon. 1. label vials 2. sterilize needle, touch colony, take cap off with pinky, sterilize, stab to bottom of tube and streak slant on way out, sterilize, cap and sterilize needle 3. repeat for all organisms

Results- Lab 5 pt.1

salmonella shigella- - non; colorless, positive; pink and acid, H2s will make black colonies macconkey- selective and differentiative - lactose; dec pH= pink - fast lactose= hot pink -peptone= alkaline->yellow/translucent - E coli growth and media around is pink EMB-metallic green (fast/fecal coliform), blue black or pink = lactose positive or negative is colorless /same color as background media methyl red and VP -sacchrolytic (glucose and one pathway) - most + M and - VP or -M and +VPO or neg for both because some other ferm pathways - asachrolytic no fermentation pathway - rare positive for both because two sets of enz ( mixed acid fermentation and 2.3 diol ferm) lysine- opp trends with LDA and LDC ODC- += purple K -= yellow mimics butt of lysine agar in. terms of acidity H2S- most likely produce is TSIA - some dont produce consistently in any of these three media carbs - oxidation dec pH -fermentation dec pH amino acids; modify environment when conditions not ideal for them - deamination dec pH -decarboxylation inc pH proteins inc pH

Nitrate Test

single enzyme test Some bacteria can reduce Nitrate (NO3 - ) to Nitrite (NO2 - ). -Some bacteria can reduce Nitrate (NO3 - ) to Nitrite (NO2 - ) and then reduce Nitrite (NO2 - ) to Nitrogen (N2). -others ignore completely The Nitrate Test aids in differentiation between genera and, in some cases, between species within a genus. The test can be performed using Nitrate Broth, a Nitrate Agar slant, or by placing a Potassium Nitrate (KNO3) impregnated disc on a freshly streaked agar plate. We use Nitrate Broth because best results in lab There are 3 possibilities: If the organism does not produce Nitrate (NO3 - ) Reductase, it will grow and ignore the Nitrate (NO3 - ). If the organism produces Nitrate (NO3 - ) Reductase, it will reduce Nitrate (NO3 - ) in the medium to Nitrite (NO2 - ). If the organism produces both Nitrate (NO3 - ) Reductase and Nitrite (NO2 - ) Reductase, it will reduce Nitrate (NO3 - ) in the medium to Nitrite (NO2 - ) and then reduce the Nitrite (NO2 - ) to other nitrogenous compounds. • Many of the bacteria that produce both Nitrate (NO3 - ) Reductase and Nitrite (NO2 - ) Reductase will completely reduce the Nitrate (NO3 - ) to Nitrogen gas (N2) If the organism does not make Nitrate (NO3 - ) Reductase, the Nitrate (NO3 - ) remains unchanged in the medium. The possible enzymatic reactions are: After incubation, we have 3 possibilities: -There is Nitrate (NO3 - ) in the medium, -There is Nitrite (NO2 - ) in the medium [all the Nitrate (NO3 - ) has been reduced], -There is Nitric Oxide (NO) or Nitrogen (N2) in the medium, probably some in the Durham Tube [all the Nitrate (NO3 - ) and Nitrite (NO2 - ) have been reduced. We have 2 reagents that when added to the medium, will react with Nitrite (NO2 - ) to produce a red color: Nitrate Reagent A contains Sulfanilic Acid, Nitrate Reagent B contains α-Naphthylamine.

Motility Test Medium

• A semi-solid medium used to determine if an organism is motile or non-motile. • Contains: • 0.5% to 1% agar. ( was less solid - semi-solid so motile can swim through medium) • We use an MTM formulation with Tetrazolium Salt. Motile organisms have flagella and can swim through the medium, out to the sides of the tube. Non-motile organisms do not have flagella and stay where they are put. Note: Obligate aerobes (organisms that require O2 for growth) can only grow where O2 is present - on the surface of the medium and, sometimes, part way down the stab • Most organisms will reduce the Tetrazolium Salt turning it red where they are growing. Turbidity (cloudiness) is evidence of growth even if the medium has not turned red. • An organism that is motile will grow out into the surrounding medium. It will migrate out away from the stab line.*** very clear line with no dispersal • In the negative tube it is easy to see that there is growth at the stab line and the surrounding medium is clear (no growth there = non-motile) • In the positive tube the medium is more turbid indicating growth throughout the medium. • If the needle did not go perfectly straight in and straight out, you may see a band of growth with clear medium around it. MTM does not fit the definition of a Differential medium because pretty much every organism will reduce the Tetrazolium Salt to some extent. Movement of the organism is not due to use of a substrate. 1. sterilize loop 2. touch solid media on colony 3. grab cap in pinky, sterilize tube, stab media straight down in center to bottom and back out 4. resterilize tube, cap, sterilize loop

• The goal would be for you to get started using the microscopes and realize just how small bacteria are. • You need to use the 100X Objective lens (1000X total magnification) to make useful observations of bacteria.

• An organism will appear 10 times larger under the 100X Objective lens (1000X total magnification) than under the 10X Objective (100X total magnification). • You want to start (trust me on this) with the 10X Objective lens because it is easier to find your specimen under lower power

Endospore Stain

• Bacteria (+ aerobic) in the genera Bacillus, Clostridium ( obligate anaerobes), and Clostridioides produce endospores** . • The infamous C. diff (Clostridium difficile) has been renamed Clostridioides difficile. *dormant phase Components of the Stain - Primary stain: Malachite Green (heat is used to help the dye penetrate the bacteria) - Counter stain: Safranin (to stain the vegetative bacteria pink) • Endospores are expected to appear green against red vegetative bacteria. We generally get poor results with this technique in our laboratory. Procedure 1. Prepare heat-fixed smear of Bacillus subtilis 2. Place the slide on the staining rack. 3. Cover the smear with filter paper. Saturate the filter paper with malachite green staining solution. Heat over a Bunsen burner till steam appears. Once steam appears, set aside till the steam disappears. Repeat for a total time of about 5 minutes. Do not allow the slide to dry out and avoid excess flooding! 4. Remove the slide, let it cool, and rinse with water for 30 seconds. 5. Rinse with water for 5 seconds to wash green out of vegetative and not spores 6. Counterstain with safranin for 1 minute. 7. Rinse with water for 5 seconds. 8. Blot dry with bibulous paper and examine under oil immersion. The bacteria with endospores will appear with either staining defects or with endospores greenish in color, while vegetative cells appear red (Figure14). A Gram stain of bacteria with endospores will show up with filling defects (Figure 15)

Atmospheric Requirement & Carbohydrate Metabolism

• Obligate Aerobes ►Respiration because O2 • Obligate Anaerobes ►Fermentation • Facultative Anaerobes ►Respiration and /or Fermentation dep on conditions etc

Oxygen Requirement

• Obligate aerobes require oxygen as the final electron acceptor of the electron transport chain. • Obligate anaerobes cannot tolerate oxygen and use an electron acceptor other than oxygen. • Facultative anaerobes can maintain life via fermentation or anaerobic respiration, though their metabolic efficiency is often reduced in the absence of oxygen. • Microaerophiles require lower than atmospheric levels of oxygen (pO2 2 - 4%). Microaerophiles cannot survive in a normal atmospheric oxygen environment (pO2 20%). • Capnophiles are microorganisms which thrive in the presence of high concentrations of carbon dioxide

pH

• Organisms are sensitive to changes in acidity because hydrogen ions (H+) and hydroxyl ions (OH- ) interfere with hydrogen bonding within the molecules of proteins and nucleic acids. • Most bacteria and protozoa are called neutrophiles because they grow best in a narrow range around a neutral pH, between 6.5 and 7.5. • By contrast, other bacteria and many fungi are acidophiles and grow best in acidic environments where pH can range as low as 0.0 (stomach acid is pH 2.0). • In contrast, alkalinophiles live in alkaline soils and water up to pH 11.5 or higher.

MacConkey Agar

• Selective/Differential Medium - used primarily to differentiate organisms in the family Enterobacteriaceae. gram - indicator • Selective: • Bile Salts and Crystal Violet inhibit the growth of Gram + organisms and most non-enteric Gram - bacteria (family Pasteurellaceae). • Differential: • Lactose - Potential substrate • Neutral Red - pH Indicator organisms that can grow- • Lactose-fermenters - Bacteria will produce acid byproducts ---►↓pH giving rise to pink/hot pink or red colonies. - Fast lactose fermenters will produce hot pink colonies with bile precipitate in the medium (the medium around the colonies turns hot pink). • Non lactosefermenters • - Bacteria will appear as colorless or translucent colonies

Salmonella-Shigella (SS) Agar

• Selective/Differential medium used for identification of bacteria in the family • Enterobacteriaceae. • Originally intended to allow differentiation of Salmonella ser. from Shigella spp. Selective: • Brilliant Green dye and Bile Salts - Inhibit the growth of Gram pos. organisms and most non-enteric Gram neg. organisms (family Pasteurellaceae). • Differential: • Lactose - Potentialsubstrate • Neutral Red - pH Indicator • Sodium Thiosulfate - Source of sulfur for Hydrogen Sulfide (H2S) production. • Ferric Citrate - Indicator of H2S production - reaction forms FeS, a black precipitate. Even one black centered colony is evidence of H2S production. - organisms that can grow • Non Lactose Fermenters - Bacteria will form colorless colonies, the medium usually turns more yellow. • Lactose Fermenters - Bacteria will form pink to red colonies. • H2S Producing Bacteria - Bacteria will have black centered colonies. procedure- 1. streak for isolation 2. draw quadrants/label 3. use loop or needle to streak but could tear surface 4.sterilize, touch, streak, repeat for each new quadrant grabbing from agar and not from original culture 5. repeat for other 3