Lab quiz 2

Pleomorphism

- variable cell morphology depending on age of cell and environment (nutrients, temperature, etc.)

Oxygen

If at any time during this process something catches fire, it is important that you extinguish it calmly and safely. The fastest way to put out a small fire is to limit its ______.

Negative staining

View morphology and arrangement without heat fixation -Benefit = less distortion of natural cells -Specimen appears light in dark background -(-) charged chromogen will not bind cell membrane can use needle OR smallloopful of specimen to nigrosin

Acid fast cell

hot pink

Solid media

may be prepared as a slant, a deep or a plate.

Negative motility

test is indicated a solid red stripe at the stab line only (middle)

Coccobacillus

- oval shape (plural = coccobacilli)

Spirillum

- rigid spiral structure

Coccus

- round (plural = cocci)

simple stain

Unstained bacterial cells are nearly transparent when observed using light microscopy and are difficult to see, even at higher magnifications. There is little contrast between the cells and the bright background when using a brightfield microscope. We can greatly improve this contrast by using a variety of staining methods. By carefully applying samples of our bacteria to a slide and then staining the specimen using dyes mixed with water or ethanol, we can observe the basic shape and arrangement known as bacterial morphology. The use of a single dye to stain a bacterial smear is termed a

Aseptic techniques

are designed to allow for safety in the microbiology laboratory. These techniques prevent laboratory workers from being contaminated by microbial cultures, prevent the cultures from being contaminated by lab workers or the environment and prevent the environment from being contaminated as well. Aseptic technique refers to a wide range of practices implemented to minimize the risk of unintentional contamination. In microbiology it is important to ensure that the only microbes growing on a medium are those that we intended to grow there. A contaminated culture occurs when unknown organisms contaminate the culture. These contaminants may come from the air carried on dust particles, flakes of skin, unsterilized inoculating loops, or anything else coming into contact with the culture that are then inadvertently introduced, often via poor aseptic technique. In research, it is often critical to avoid contamination of samples, cultures, etc., so that an experiment isn't ruined. In health care, protecting patients from infection will be a major concern, in addition to identifying the bacterium that has caused your patient's infection, not the normal microbiota from your mouth that contaminated the specimen.

Endospores

are metabolically inactive and are resistant to heat, desiccation, chemicals and radiation. When environmental conditions become more favorable, the endospores may germinate into metabolically active vegetative cells.

Inoculating Needles

are most often used to perform a technique known as a stab inoculation. You will learn this technique in Lab Module 06 when we analyze bacterial motility.

Inoculating Loops

are often used in the lab setting to transfer specimens from various types of liquid and solid culture media. You will be learning how to properly do these transfers in each of the experiments found in Module 04.

Agar slants

are often used to store or transport pure cultures. A benefit of storing a culture on a slant is that there is minimal risk of contamination or of losing the culture due to the medium drying out. Slants can be stored for weeks, even months, especially if refrigerated.

Semi-solid deeps

are sometimes used and in this case, a lesser percentage of the solidifying agent agar is added.

Bacillus Clostridium

are two examples of endospore forming bacteria

Wet mounts

are used for viewing living specimens in a more natural state. This quick and easy technique simply requires a drop of sample in a liquid environment to be placed on a slide.

V-shape

bacilli that are arranged at an angle

Non acid fast cell

blue

Mixed cultures

cannot be separated in broth media

Streptococcus

chain of cocci

streptobacillus

chain of rods

Lophotrichous

cluster of flagella at one end

General-purpose or all-purpose media

contain a variety of nutrients that will facilitate the growth of a wide range of microbes. An example of general purpose media is tryptic soy broth or a tryptic soy agar (TSA) plate

Enriched media

contain additional nutrients to support the growth of a wide variety of organisms, including some that are more fastidious or difficult to grow. The media are enriched usually by adding blood, serum or egg. Examples of enriched media include blood agar (right).

Cheek cell

eukaryote

Selective media

favor the growth of one type of organism while preventing or inhibiting growth of others. Selective inhibition can be achieved many different ways. Antibiotics, salts, dyes, sugars or other specific inhibitors may affect metabolism or enzyme function of some organisms and therefore their growth. For example, phenylethyl alcohol agar (PEA) is selective for Gram-positive bacteria. It inhibits most Gram-negative bacteria by interfering with DNA synthesis. Clinically, PEA agar is used to isolate Gram-positive Staphylococcus spp. and Streptococcus spp. from mixed bacterial culture specimens.

Amphitrichous

flagella at both poles of the cell

Peritrichous

flagella distributed over the entire cell

Gram-negative bacteria

have a much thinner layer of peptidoglycan within the periplasm of their cell wall. Additionally, Gram-negative bacteria have an outer membrane that is external to the peptidoglycan layer that contains lipopolysaccharide (LPS). Gram-negative bacteria will lose the crystal violet during the decolorization step to become colorless when the LPS layer dissolves in the presence of the alcohol. The last step of the Gram stain is to counterstain with safranin. Gram-positive bacteria will stain purple because they retain the primary stain, crystal violet. Gram-negative bacteria will appear the same color as the counterstain, reddish-pink.

Gram-positive bacteria

have a thick peptidoglycan layer made up of many (up to 40) layers of alternating carbohydrates (N-acetylglucosamine and N-acetylmuramic acid) and small peptides. This thick cell wall prevents these microbes from losing the purple stain upon decolorizing.

Staphylococcus

irregular clusters(number of cells varies)

Motility medium

is a semisolid agar deep containing TTC (triphenyltetrazolium chloride).

Mannitol salt agar (MSA)

is another example of a medium that is both selective and differential. MSA agar (left) favors the growth of organisms that can tolerate the 7.5% NaCl in the medium. These bacteria are said to be halotolerant. MSA differentiates based on the ability of the bacteria to ferment the sugar mannitol. Fermentation of mannitol produces acid, which lowers the pH in the medium. The pH indicator, phenol red, is and orange-red color at a neutral pH and turns yellow when it is acidic (S. aureus).

Sterile Swabs

may be used to collect samples for transfer to petri dishes containing solid culture media. Environmental sampling often utilizes swabs for this type of analysis, similar to what was done to study Microbial Ubiquity in Experiment 2.1. Swabs are also used in the clinical setting to obtain patient samples (e. g. throat cultures) for culturing and diagnosis.

triphenyltetrazolium chloride

motility medium

No motility

right B

palisade

side by side rods

montrichous

single flagellum at one end

Gram positive bacilli

some _____ _______ _______ have the ability to produce intracellular structures known as endospores. An endospore is a highly resistant form of a cell that protects the organism from unfavorable environmental conditions.

Broth

which is depicted in the image above. Broth is used primarily to grow and maintain bacterial cultures.

Colored by dye.

Appearance of Positive staining cell

blood agar

The media are enriched usually by adding blood, serum or egg. Examples of enriched media include :

Normal

control

Dried stain

or slide not rinsed properly. Size variability = clue

bacillus

- longer than wide (plural = bacilli)

Vibrio

- bent rod (comma shape)

Spirochete

- flexible spirally twisted structure

acid fast stain

-Although the great majority of bacterial cells may be stained with the Gram stain, there are a few genera that have a unique cell wall which prevents utilizing this method. These bacteria, such as those in genera Mycobacterium and Nocardia, have a waxy substance called mycolic acid in their cell wall which prevents the aqueous Gram stain reagents from entering the cells. Acid-fast organisms are also highly resistant to desiccation and disinfectants due to this waxy chemical. Because the cell wall is so resistant to most compounds, heat is used as a mordant to drive the primary stain, carbol fuschin, into the cell wall. When the smear is rinsed with an acid alcohol decolorizer, the stain is removed from all non-acid-fast cells, but the acid-fast organisms retain the dye. We then counterstain with methylene blue and the decolorized cells will appear blue. -Examples of medically important pathogens that are acid fast include those that cause diseases such as tuberculosis and leprosy.

Capsule stain

-Capsule is mucoid and composed of polysaccharides -Well-organized layer, not easily washed offFunctions as a virulence factor -Makes bacteria less vulnerable to phagocytosis/digestion

Lab safety

-Keep your work area clean and uncluttered -Decontaminate the work surface with a germicide before and after completing an experiment -Sterilize all media before use (this will have already been done for you.) Use sterile instruments for transferring specimens. -Incinerate or Flame-sterilize inoculating loops and needles before and after subculturing. Wait until you are ready to complete the procedure before opening the lid of an agar plate or removing the lid from a tube or flask. -Heat the mouth of glass tubes, flasks or bottles to create an updraft that minimizes the risk of pulling air into the container. -Do not take the lid off of a plate or tube and set it down on the work surface. Work quickly to minimize exposure to the environment. -Avoid talking, singing, whistling, etc. when you have a culture container open to avoid contamination from aerosols In our lab you will usually sterilize the loop and other instruments in an incinerator (left) or, for certain procedures, in the flame of a Bunsen burner.

Flagella stain

-Mordant on flagella -Carbol fuchsin simple stain

differential stain

-Most widely used staining procedure in bacteriology -Differentiates based on cell wall properties -Purple = Gram positive -Pinkish/Red = Gram negative -Crystal violet(primary stain) -Iodine(mordant) -Alcohol(decolorizer) -Safranin(counterstain)

Capsule stain

-Some bacteria produce a thick polysaccharide capsule external to the cell wall. Capsules are virulence factors that protect bacteria from phagocytosis by hiding their "red flags" that tell our white blood cells to attack. When organisms produce these capsules, they can colonize the body tissues without identification. You will learn of many diseases caused by bacteria that evade the immune system in just this way. Streptococcus pneumoniae which causes classic bacterial pneumonia is an example of a capsule forming organism. -The capsule stain, shown here, is an example of a combined positive stain and a negative stain. It first employs a positively charged basic dye to stain the cells while using heat to force the dye into the cell across the capsule. This is followed by a negatively charged, acidic dye which stains the background leaving clear capsules unstained. One thing to keep in mind is that environmental factors such as temperature, the medium in which the culture is grown and the age of the culture will affect capsule formation.

Bunsen burner

-Sometimes, an open flame (Bunsen burner) and alcohol will be used for decontamination, for example, when sterilizing forceps or a bent glass rod (see below). This description will use forceps but a bent glass rod will be sterilized in similar fashion. In this case you will do the following: 1.Locate the Bunsen burner, beaker of 70% isopropyl alcohol, and the forceps. 2.Light the Bunsen burner when ready to work (never leave flame unattended).CAUTION: Alcohol is highly flammable. Take extra care to position the alcohol far out of the way from the Bunsen burner. 3.Hold the forceps in your dominant hand. Dip carefully into the isopropyl alcohol for 10 seconds. -Once you've put the forceps in the alcohol, keep it angled down so that no alcohol drips back onto your hand. 4.Without touching the forceps to anything, carefully place it in the flame of the Bunsen burner. -Hold the forceps in the flame of the burner. Usually it will catch on fire as the alcohol is flammable. DO NOT PANIC! This is supposed to happen. Just exercise extreme caution. Again it is very important to point the forceps down as the alcohol and the flame could burn your hand. -Allow the forceps to cool for about 15-20 seconds before using

endospore stain

-can be used to differentiate Gram-positive bacilli (i.e. B. anthracis, B. subtillis, C. tetani, C. botulinum, C. difficile) that are endospore forming from non-endospore forming Gram-positive bacilli such as Lactobacillus and Corynebacterium. -Although not all endospore formers are pathogenic, still the medical importance of endospores cannot be overstated. Many diseases can be caused by the transmission of endospores present in the soil or in improperly canned foods. Examples of clinically important diseases include anthrax, tetanus and botulism.

stab inoculation

-is performed using an inoculating needle containing the specimen of interest and is then incubated. -Observation of the area of growth can then be done to evaluate motility. TTC is converted to an insoluble red pigment by the bacteria as they grow to make visualization easier.

Quadrant streak plate method

1. If desired, draw four quadrants on the bottom of the plate and label them 1 through 4. 2. Sterilize the loop, and let it cool for about 15 seconds. Aseptically remove a loopful of the bacterial mixture from the stock culture. a. For pure culture slants, use the loop to gently scrape a small amount of bacteria off the surface of the agar without digging into the agar. b. For the mixed culture broth, be careful to mix the culture before aseptically obtaining the loopful of bacteria. 3. Streak out the loopful of bacteria onto the agar completely covering quadrant 1. Be sure to spread the bacteria on the agar from edge to edge within the quadrant. 4. Remove the inoculating loop from the plate and sterilize it in the incinerator or open flame to kill any remaining bacteria. Allow the loop to cool for about 15 seconds. 5. Rotate the plate a little less than 90o. Insert the loop under the lid and briefly touch it to an open area of the agar in section 2. Drag the loop over the bacteria in quadrant 1 and then streak the bacteria from quadrant 1 into quadrant 2. Do not go back into the first streak more than three times. 6. Remove the loop, sterilize it, and let it cool as before. Rotate the plate a little less than 90o. Insert the loop under the lid and briefly touch it to an open area of the agar in section 3. Drag the loop over the bacteria in quadrant 2 and then streak the bacteria from quadrant 2 into quadrant 3. Do not go back into the second streak more than three times. 7. Remove the loop, re-sterilize it, cool it in the agar as before, and repeat the streaking process into quadrant 4. 8. Invert the plates (lid side down) and incubate as directed by your instructor.

wet mount procedure

1. Place small drop of water on clean slide 2. Using sterile technique place a loopful of culture in center water on slide 3. Place cover slip over slide 4. Absorb excess water

Endospore stain- Schaffer-Fulton method

1.Begin with a heat-fixed emulsion 2.Cover the smear with a strip of bibulous paper -Apply Malachite Green stain -Steam for 5 minutes -Keep the paper moist with stain. -Perform this step with adequate ventilation and eye protection

T streak method

1.Draw a letter "T" on the bottom of the plate and label the sections 1-3. Keep plate on the bench agar UP, lid DOWN so that you can see your sections. 2. Sterilize the loop and let it cool for about 15 seconds. Aseptically remove a loopful of the bacterial mixture from the stock culture. a. For the pure culture slants, use the loop to gently scrape a small amount of the bacteria off the surface of the slant without going into the agar. b. For the mixed culture broth, use as usual but be very careful to mix the culture before obtaining the loopful so that both organisms will grow. 3. Pick up the plate out of the lid and hold at an angle to avoid airborne contaminants. 4. Streak out the loopful of bacteria onto the agar plate into section 1 following the procedure introduced above and place the plate back in the lid. 5. Sterilize the inoculating loop to kill any remaining bacteria on the loop and cool for about 15 seconds. 6. Rotate the plate so that section 2 is available for use. Remove the plate from the lid, insert the loop at the edge of the agar near section 1. Carry over bacteria from section 1 three times and then streak out the bacteria picked up into section 2. Remember to NOT go back into pattern 1 after the three carry-overs. Place the plate back in the lid and turn the plate again. 7. Sterilize the loop, cool it, and repeat the streaking process into section 3, carrying bacteria over from section 2 three times. Place plate back in the lid. 8. Invert the plates (lid down) and incubate as directed by your instructor.

aseptic technique for test tube to an agar plate

1.Follow steps 1-7 above. Note: You will only have the original culture tube in the non-dominant hand. 2.Once the inoculum is on the inoculating loop, carefully tilt the lid of the agar plate above the plate so that you can get the inoculating loop between the lid and the plate. The lid should cover the agar surface as completely as possible at all times in order to avoid contamination. Do not rest the lid on the lab table or completely expose the plate of media to the air to avoid possible contamination from air-borne contaminants. Insert the inoculating loop of bacteria and spread it over the plate as directed by your instructor. Do not dig into the agar; simply let the tip of the loop rest gently on the surface of the agar and move it across the surface of the agar. 3.Sterilize the inoculating loop after the bacterial transfer. 4.Incubate the plate as directed by your instructor. Be sure to invert the plate so that the lid is on the bottom (agar UP) to avoid possible condensation from the inside of the lid dripping onto the medium you have just inoculated with the bacterial culture.

aseptic technique for handling test tubes

1.Have all needed test tubes of media available at your lab bench. Make sure that all test tubes are appropriately labeled as directed by your instructor. Do not write directly on the test tube cap. Be sure that the label does not interfere with you being able to see the inside of the tube. 2.Obtain your stock culture tube from your instructor, making sure that you always transport it in a test tube rack. You should never carry a test tube containing bacteria by hand (see the safety rules from Module 01). Tubes should never be laid directly on the lab bench; the tube should be in a test tube rack or held in your hand at all times. 3.Hold the inoculating loop in your dominant hand like you hold a pencil. Hint: Actually pretend it is a pencil and practice holding it as if you plan to write with it. 4.Sterilize the inoculating loop by placing it into the bacti-cinerator until the loop turns an orange color (3-5 seconds). Do not leave it in the bacti-cinerator when you are not using it, as the wire can melt and damage the bacti-cinerator over time. You also could cause a fire. 5.Pick up the culture tube in the middle of the tube (not the cap) with your non-dominant hand, 6.If the test tube with the bacterial culture is a broth, you will need to gently rub the tube between the fingers of both hands to mix the bacteria before you do step 5. Be very careful when you do this so that you do not accidentally drop the tube. 7.Hold the culture tube at a slight angle to avoid airborne contaminants. 8.Grasp the cap of the culture tube with the little finger and ring finger of the hand holding the inoculating loop and remove the cap, pulling it away from the culture tube. Hold the opening of the tube close to the bacti-incinerator for 3-5 seconds. 9.Carefully place your loop into the bottom of the culture tube to obtain a loopful of specimen. Avoid touching the side of the tube. Again, hold the opening of the tube close to the bacti-incinerator for 3-5 seconds. Re-cap the tube and place in your test tube rack. 10.Pick up the tube you wish to inoculate and repeat steps 6-9. Place tube the bacteria from the inoculating loop to the test tube of media being inoculated. For a broth tube, gently shake the inoculating loop of bacteria in the new test tube of media. For an agar slant tube, gently steak the surface of the media in the new tube using a zig zag pattern. 11.Carefully sterilize the inoculating loop after the bacterial transfer. 12.Follow the same procedure listed for all bacterial transfers from test tube to test tube. 13.After bacterial transfers, incubate the tubes as directed by your instructor.

Quadrant streak plate method

1.Label the bottom of the Petri dish of media as directed by your instructor. Also, draw four quadrants on the bottom of the plate. You might want to label the quadrants 1 - 4, as well. 2.Aseptically remove a loopful of the bacterial mixture from the stock culture. 3.Streak out the loopful of bacteria onto the agar plate into quadrant 1 following the procedure introduced above. 4.Remove the inoculating loop and sterilize it to remove any remaining bacteria on the loop. Insert the loop under the lid and cool it at the edge of the agar near quadrant 1. 5.Rotate the plate so that quadrant 2 is available for use. Carefully tilting the lid up again, cross over the streaks in quadrant 1 and streak out the bacteria picked up into quadrant 2. 6.Remove the loop, re-sterilize it, cool it in the agar as before, and repeat the streaking process into quadrant 3. 7.Remove the loop, re-sterilize it, cool it in the agar as before, and repeat the streaking process into quadrant 4. 8.Incubate the plates as directed by your instructor.

Negative staining procedure

1.Palce a single drop of Nigrosin or India ink near one end of a slide. Use a needle to disperse a small amount of organism in the dye. 2.Place a second slide in front of the drop and move the slide backward until it touches the dye, speeding the dye across the trailing edge of the spreader slide. 3.Slide the spreader slide forward, dragging the suspension of organisms across the slide. The should result in a smear that is quite thick at one end and very thin at the other. 4.After allowing the slide to air dry, it may be examined under oil immersion.

Diplococcus

2 cells

Vegetative spores

24h Bacillus cereus

Tetrad

4 cells

Central elliptical spores

48h Bacillus cereus

Free spores

72h Bacillus cereus

differential stain

A staining procedure using more than one dye is termed a _____ _____. As the names suggest, a simple stain is easy to do but gives us limited details about our specimen. A differential stain is a bit more complicated, but gives us important information about structural differences that may be very useful in identifying an unknown organism. In this module we will introduce properly applying bacteria to a slide, simple staining of the cells or the background, and the most important differential stain used in bacteriology. Before staining the cells using a positive stain, bacteria are carefully applied to a slide. This is known as a bacterial smear. In this module, you will learn how to make smears from a variety of different sources.

Slant culture preparation

Agar slants are often used for pure culturing, particularly when we need a higher density of cells than can be supported in a broth culture. Additionally, if we wish to store our cultures for extended periods of time under refrigerated conditions, cultures can be maintained safely in a slant. In this experiment we will transfer bacteria from a broth culture to a sterile slant to create a pure slant culture. Lab 01 1. Prepare your work area and turn on your incinerator and allow it to heat up before use. 2. Each student will obtain the broth culture you wish to subculture and a new agar slant. Label the slant with specimen name and date. 3. Sterilize the inoculating loop by placing it into the incinerator (open flame) until the loop turns an orange color (~5 seconds). Cool loop for about 15 seconds before subculturing bacteria. 4. Pick up the broth culture tube in the middle of the tube (not the cap) with your non-dominant hand. Gently mix to resuspend the bacteria. Hold the culture tube at a slight angle to avoid airborne contaminants and be careful not to spill. 5. Grasp the cap of the culture tube with the little finger and ring finger of the hand holding the inoculating loop and remove the cap, pulling it away from the culture tube. Hold the opening of the tube close to the incinerator for 5 seconds. 6. Carefully place your loop into the broth culture to obtain a loopful of bacteria. Avoid touching the loop to the sides of the inside of the tube. Again, hold the opening of the tube close to the incinerator for ~5 seconds. Re-cap the tube and place it in your test tube rack. 7. Pick up the slant you wish to inoculate in your non-dominant hand. Hold the culture tube at a slight angle to avoid airborne contaminants. 8. Grasp the cap of the slant tube with the little finger and ring finger of the hand holding the inoculating loop and remove the cap, pulling it away from the culture tube. Hold the opening of the tube close to the incinerator for about 5 seconds. 9. Gently steak the surface of the new agar slant with the loop using a zig zag pattern from bottom to top. 10. Hold the opening of the tube close to your incinerator for 5 seconds, re-cap it and place it in your test tube rack. 11. Sterilize the loop in the incinerator or open flame. 12. Incubate your slant culture as directed by your instructor.

tryptic soy agar (TSA) plate

An example of general purpose media is tryptic soy broth or a -We will use this media most often for our culturing needs.

colony forming unit, CFU

Any cell (hereafter referred to as a _________ _________ _______)supported by the media and designated environment can produce an isolated colony of daughter-cells through binary fission. In spite of the rapid mutation rates within bacterial communities, this cell-group is generally regarded as pure. Harvesting and re-streaking this population consequently ensures that subsequent work involves only a single bacterial species.

Clear and colorless

Appearance of negative staining cell

heat fixed smear

Before staining and observing a specimen under the microscope, a heat fixed smear is often prepared. The goal is to carefully prepare a thin, uniform film of bacteria on a glass slide that will stain evenly throughout the specimen. When we prepare a smear from a liquid (broth culture, blood or urine), we remember that there are usually relatively few bacteria in the sample and aim to concentrate those organisms on the slide so they will be better viewed. Placing one or two loopfuls of culture on our slide in a concentrated area usually is best. When we prepare a smear from a solid (agar slant or plate), we keep in mind that there are many, many more organisms in this sample. We need to dilute our specimen by adding a very small amount from the agar culture to a liquid on the slide (water) and spreading it evenly on the surface of the slide using our loop. Once smears are prepared, they must be completely air dried. This prevents cells from being distorted or bursting when exposed to heat. When smears are dry, they are cloudy and no moisture is seen on the slide. The final step in smear preparation is heat fixing the cells. Heat fixing a smear accomplishes three very important things. It helps to prevent cells from being washed off during the staining procedure by melting the cell protoplasm and causing it to "stick." It also disrupts the cell wall slightly so that the bacteria will better absorb the dye. Lastly, heat fixing may kill the bacteria, although careful handling of slides is always recommended. Heat fixing may be done by passing the slide just above an open flame 3-4 times or (in our lab) by holding the slide on the surface of the bacti-cinerator for 45 seconds. Note: care should always be used when using a flame or incinerator to prevent burning. Please review Fire Hazard Safety in Module 01. You will prepare a variety of heat fixed smears in experiment 1 in preparation for simple and differential staining of the cells.

Broth Culture Preparation

Broth cultures are often used for general bacterial culture growth and maintenance. In this experiment we will transfer pure colonies to sterile broth media using aseptic techniques to create pure culture broths. Lab 01 1. Prepare your work area and turn on your incinerator to heat up before use. 2. Each student will obtain a test tube rack and one new broth tube. Label the tube with specimen name, your initials and date. Always record in your lab notebook the name of the specimen you are using, and the culture media being used as well. 3. Identify an isolated colony on the Petri plate you wish to subculture into the broth. 4. Sterilize the inoculating loop by placing it into the incinerator or passing it through an open flame until the loop is red-hot (about 5 seconds). Cool the loop for about 15 seconds. 5. Hold the plate at an angle, and slightly lift the lid using clamshell technique to avoid airborne contaminants. 6. Use the loop to gently scrape the colony off the agar. Be careful not to gouge the agar and only touch the one colony. Close the plate and set it down on the bench. 7. Carefully pick up the sterile broth in the middle of the tube (not by the cap) in your non-dominant hand. Holding it at an angle, remove the cap with the pinkie of the hand holding the loop and place the opening of the tube close to your incinerator for about 5 seconds. 8. Carefully place your loop containing the colony into the broth without touching the sides of the inside of the tube and mix gently with your loop. 9. Remove the loop from the tube without touching the sides. 10. Again, hold the mouth of the tube close to the opening of the incinerator for 5 seconds. Cap the tube and place it in your test tube rack. 11. To sterilize the loop, place it into the incinerator (or flame) until the wire turns an orange color (~5 seconds) 12. Incubate your broth culture as directed by your instructor.

Basic stains (most)

C+ ionically binds (-) charged cell membrane

Acidic stains

C- binds to (+) charged structures/ repels (-) charged cell membrane

Klebsiella pneumoniase

Capsule stain

Motility medium

Clean stab! -Tetrazolium salts turn red when reduced Diffuse growth radiating from stab = (+)Growth only along stab = (-)

Negative staining

Clear and colorless Stained(dark gray or black)(background) Acidic dyes: -Nigrosin -India ink

Positive staining

Colored by dye Background not stained (generally white) Basic dyes: -Crystal violet -methylene blue -safranin -Malachite green -Simple stains: -Differential stains: Gram stain Acid-fast stain Spore stain -Special stains Capsule Flagella Spore Granules Nucleic acid

Bacterial staining

Composition of stain (dye): -Solvent (water or ETOH) -Chromophore (pigment)

Unfiltered dye

Crystals

Acid fast stain

Differential for bacteria in genus Mycobacterium and Nocardia -M. leprae, M. tuberculosis, M. smegmatis -N. brasiliensis, N. cyriacigeorgica, N. farcinica -Mycolic acid in cell wall = waxy/lipid coat -Confers ability of bacteria to resist decolorization with ACID ALCOHOL confers acid fastness to bacterium -Due to high wax content of wall - tend to adhere to each other after cell division forming cords

Diplobacillus

double bacillus

Aseptic transfers

In this experiment you will use sterile saline to practice aseptic technique and evaluate your lab partner's technique. 1. Turn on your incinerator and allow it to heat up. 2. Each student should obtain a test tube rack, 2 sterile saline tubes, and an inoculating loop. 3. Hold the inoculating loop in your dominant hand like you hold a pencil. Sterilize the inoculating loop by placing it gently into the back of the incinerator until the loop turns an orange/red color (about 5 seconds). a. Do not ever leave the loop in the incinerator when you are not using it. The wire can melt and damage the incinerator over time. You also could cause a fire. 4. Allow the loop to cool about 15 seconds before making a transfer. 5. Pick up the sterile saline tube that is your practice-culture tube in the middle of the tube (not the cap) with your non-dominant hand. 6. Gently flick the tube and rub it between the fingers of both hands to mix, as you would if it were a bacterial broth culture. Be very careful that you do not accidentally drop the tube or spill the tube contents. 7. Holding the culture tube at a slight angle to avoid airborne contaminants, grasp the cap of the culture tube with the little finger and ring finger of the hand holding the inoculating loop. Pull the cap away from the tube. 8. Hold the mouth of the tube close to the opening of the incinerator for about 5 seconds. 9. Carefully place the cooled loop into the bottom of the saline (practice-culture) tube to obtain a loopful of "specimen." This is called an inoculum. Avoid touching the sides of the tube with the loop. 10. Again, hold the opening of the tube close to the opening of the incinerator for about 5 seconds. 11. Re-cap the tube and place it in your test tube rack. LM 04: Aseptic Technique & Pure Culturing 12. Pick up the tube you wish to inoculate. Holding the tube at a slight angle to avoid airborne contaminants, grasp the cap of the culture tube with the little finger and ring finger of the hand holding the inoculating loop. Pull the cap away from the tube. 13. Hold the mouth of the tube close to the opening of the incinerator for about 5 seconds. 14. Place the loop with the inoculum into the saline of the tube being inoculated. Gently mix the loop in the saline. Again, avoid touching the sides of the tube with the loop. 15. Hold the opening of the tube close to the opening of the incinerator for about 5 seconds. Re-cap the tube and place it in your test tube rack. 16. Re-sterilize the inoculating loop after the transfer by holding it inside of the incinerator until the wire is red-hot. 17. Have your partner observe and critique your technique at each step. Then, let your partner practice the transfer while you critique. 18. Repeat several times until you feel reasonably comfortable and ready to transfer real bacterial cultures.

Motility

Left A

Antiseptic transfer

Mix broth cultures before sampling by gently tapping side of the tube or rolling it between your fingers. Hold the tube at an angle to minimize airborne contamination. Remove cap with little finger - DO NOT set the cap down on bench. Quickly pass the tube through inner cone (2-3X). This creates a vacuum pulling air out of the tube, minimizing the risk of contamination. Dip the loop into the broth culture. Be sure the loop has cooled - if it sizzles when placed in the culture, it was too hot! Be careful not to touch side of the tube or the lip with the unsterilized handle of the loop. Flame the mouth of the culture tube 3-4 times before replacing the lid and place tube in your test tube rack. Quickly transfer the culture on the loop to the new slant or broth. If subculturing to a broth: Remove lid, flame mouth, place tip of the loop in new broth culture. If subculturing to a slant: Remove lid, flame mouth, insert loop to base of the slant. Pull the loop inn a Zig-zag pattern. If subculturing to a plate: Return tube to rack (after re-flaming lip). Lift the lid slightly, use it as a shield. This is called the "clam shell technique." Streak or spot the culture on the agar plate. DO NOT remove the lid and set it on bench, as this will increase risk of contamination. Note: There are many ways to inoculate a plate and these will be described by your instructor. Be sure to flame the loop after subculturing to any type of media.

wet mount

Motility -True movement with random runs and tumbles Brownian movement -Motion exhibited by minute particles of matter when suspended in a fluid

Stained (dark gray or black)

Negative staining background

simple staining

One dye used to enhance observation of microbe morphology and arrangement Stain: -Carbol fuschin (~20 sec) -Safranin (~45 sec) -Crystal violet (~45 sec) -Methylene blue (~1 min)

in vivo culture

Organisms use a variety of nutrients to meet their energy needs and to provide materials required to build organic macromolecules and cellular structures. When microorganisms are growing in another organism like an experimental animal (or us), this is called -The living host provides the necessary nutrients to support the growth of the microbes. When we culture the organisms in the lab, this is known as n that case, a growth or culture medium must provide these nutrients to support the growth of the microbes. The culture media provide the elements that most bacteria need for growth which include carbon, hydrogen, oxygen, nitrogen, phosphorus and sulfur. Fastidious organisms have complex growth requirements and are thus more challenging to grow in the lab.

Citrobacter diversus

Pink

Crystal violet Methylene blue Safranin Malachite green

Positive staining

Not sained (generally white)

Positive staining background

bacterial endospores

Protective non-vegetative state characteristic of some bacterial species -Clostridium, Bacillus, Desulfotomachulum spp. Spores germinate →vegetative cells

Staphylococcus epidermidis

Purple

heat fixed smear

Purpose - prepare thin, uniform film of bacterial on the slide prior to microscopic examination -Use either 1-2 loopfuls of a broth culture, or an emulsion of culture, spread evenly on slide surface. -Smear is thoroughly air-dried then heat-fixed using one of the following techniques: -Pass the slide through a flame -Use of a slide warmer -Using back of incinerator -Heating adheres smear to glass, disrupts the wall to better take on stain and kills the cells First step of most staining techniques Procedure - clean, labeled slide -Broth culture-concentrate -Solid medium-dilute -Do not blow on or wave slides in air Using dropper bottle, place two loopfuls H2O on clean slide -Aseptically remove tiny amount of culture from agar surface and mix with H2O (Broth can smear directly onto slide without water) -Using loop, spread mixture over slide to form a thin film Allow suspension to completely air dry -Heat fix -- Pass the slide (film-side up) through the flame of the Bunsen burner OR hold slide on back of incinerator (smear up) for 45 sec

Bacillus anthracis

Rod-shaped gram- positive bacteria in a cerebrospinal fluid sample stand out from round white blood cells, which also accept the crystal violet stain.

Streak Isolation of Bacterial Cultures

The first step in the identification of an unknown bacterial specimen is growing it in pure culture. In this experiment we will introduce two different streak isolation techniques to separate mixed bacterial samples into individual colonies. The goal is to use the surface area of the agar to dilute the concentration of bacteria with each streak. 1. Begin by practicing your preferred streak plate method (see below and information provided by your instructor) in your lab notebook. 2. Trace a petri plate and draw out the pattern you will streak. Number the sections of the plate (draw a T if using the T-streak method). 3. Each time you need to sterilize your loop, put a star. 4. Compare your drawing with other members of your lab and critique. Now you are ready to streak the agar plates. Lab 01 1. Prepare your work area. Be sure to turn on the incinerator to heat up. 2. Each student will obtain two agar plates and complete two streak isolations: one of a pure culture and one from a mixed broth culture. 3. Label the bottom of each plate with the date, culture name, and your name/initials. Record in your lab notebook the name of the specimen you are using, and the culture media being used. 4. Streak isolate the cultures using the method designated by your instructor

Isolating pure culture from a bacterial mixture

The streak plate method for isolating pure cultures from a bacterial mixture (T streak method): 1.Label the bottom of the Petri dish of media as directed by your instructor. Also, draw a T on the bottom of the plate. Label the sections 1-3. Keep plate on the bench agar UP, lid DOWN so that you can see your sections. 2.Aseptically remove a loopful of the bacterial mixture from the stock culture. Pick up the plate out of the lid and hold at an angle to avoid airborne contaminants. 3.Streak out the loopful of bacteria onto the agar plate into section 1 following the procedure introduced above and place plate back in the lid. 4.Sterilize the inoculating loop to remove any remaining bacteria on the loop. Insert the loop under the lid and cool it at the edge of the agar near section 1. 5.Rotate the plate so that section 2 is available for use. Remove the plate from the lid, carry over the streaks from section 1 three times and then streak out the bacteria picked up into section 2. Remember to NOT go back into pattern 1 after the three carry-overs. Place plate back in the lid and turn the plate again. Now pattern 1 should be on the bottom. 6.Sterilize the loop, cool it in the agar as before, and repeat the streaking process into section 3, carrying over from section 2 three times and place plate back in the lid. 7.Incubate the plates as directed by your instructor.

Brownian motion

This vibrational movement exhibited by non-motile organisms is known as

Decontamination of loops

To decontaminate metal inoculating loops and needles (for the transfer of bacteria) you will use an incinerator. This is a safer method of decontamination than an open flame and is recommended by the American Society of Microbiology to be used in all introductory microbiology labs by doing the following: 1.Turn on the incinerator and let it heat up until it appears red hot in the center. This should take 5-10 minutes. 2.Hold inoculating loop or needle like a pencil, in your dominant hand. Be sure you are holding it at the end as the heat will conduct. 3.Place the loop inside the incinerator so that it almost touches the back of the device for 3-5 seconds to insure decontamination and then remove from the incinerator. Do not leave a loop or needle in the incinerator for more than 5 seconds as it can catch on fire and create a biohazard in the laboratory. 4.Allow loop to cool for about 15 seconds. Now you may safely use loop or needle to transfer cultures. 5.Loops and needles should be decontaminated in this way before and after each culture transfer.

Acid fast stain

Ziehl-Neelsen Procedure -Primary stain - lipid soluble carbolfuchsin, with steam heat acting as mordant -Cells resist decolorizing by acid alcohol -Non acid-fast cells will be decolorized -Counterstain of methylene blue will stain decolorized cells Acid-fast (+) cells stain hot pink Non-acid-fast cells stain blue

differential media

allow distinctions to be made between similar organisms by targeting specific biochemical properties of microorganisms. For example, a differential medium may test for presence of enzymatic activity, or other metabolic properties of bacteria. An example of a differential medium is nitrate broth. This is used in the nitrate reduction test to determine if a bacterium possesses the enzyme nitrate reductase (nitratase), enabling it to reduce nitrate (NO3-) to nitrite (NO2-), or another nitrogenous compound. Reduction of nitrate is an important test in the identification of both Gram-positive and Gram-negative bacteria.

pure culture

contains only one species of microorganism.

sarcina

cubical packet of 8, 16, or more cells (8-64 )

Over-decolorized

dark brown cocci, not purple

fastidious

difficult to grow

streak plate technique

is based on progressive dilution of a sample (right), with the aim of separating individual cells from one another. I) The inoculum is initially dispersed using a zig-zag motion with non-overlapping streaks, creating an area with a relatively dense bacterial population. II-IV) Streaks are drawn from the preceding area, using a sterile inoculation loop each time, until the fourth quadrant is reached. V) A final zig-zag motion directed towards the middle of the plate forms a region where the inoculum has been markedly diluted, allowing colonies to appear separate from one another. The streak plate technique can also be combined with the use of selective and/or differential media. A selective medium will inhibit the growth of certain organisms (e.g. through addition of antibiotics or other inhibitors) while a differential medium will help distinguish one organism from another (e.g. through hemolysis on blood agar plates). The use of selective and differential media will be described in more detail in future modules. As in all other bacterial transfers, good aseptic technique is critical to avoid contamination and safety. The initial streak-plate may contain colonies originating from cells with different genetic makeup or (depending on sample purity) from different bacterial species (Figure A right). Through subsequent isolation of a single colony, where all units are derived from a common mother-cell, the second streaking procedure generates a relatively pure bacterial population, suitable for further characterization or inoculation into broth (Figure B right). A pure culture can be generated from a mixed sample through isolation of a single, isolated colony. A) Growth of a single bacterial cell (CFU) generated a pure colony, separated from those of other species and strains. This CFU was used for subsequent streaking onto a new plate B) A second plate, where the bacterial population consists solely of cells derived from the initial CFU. Keep in mind, that the first step in the identification of an unknown bacterial species is isolated it and growing it in pure culture.

Blood agar

is considered enrichment and differential. Not only does the blood in the agar provide extra nutrients for growth, but if an organism produces a virulence factor known as a hemolysin, it will be able to lyse the blood cells in the medium. This causes a partial (alpha) or complete (beta) clearing of the agar surrounding the colony. Non-hemolytic organisms (gamma) show no agar clearing. Analysis of hemolysis can help in the identification of an unknown organism. A throat swab showing evidence of beta hemolytic chains of cocci (streptococci) may suggest the presence of the pathogen Streptococcus pyogenes which causes streptococcal pharyngitis (strep throat).

phenylethyl alcohol agar (PEA)

is selective for Gram-positive bacteria

Agar

is typically used as the solidifying agent in this type of media. Agar is a gelatin-like substance that is extracted from red algae and is an ideal solidifying agent for a variety of reasons. Agar is: nontoxic to most microbes, physiologically inert, can't be digested or degraded by most microbes, stable to sterilization temperatures (remains solid over a wide range of growth temperatures), and it is generally transparent.

coverslip

is usually added to flatten the field. The specimen may be difficult to observe as living cells tend to be very transparent having little contrast with the bright background of our brightfield microscope. This may be improved by limiting the light directed onto the specimen by adjusting the iris diaphragm to reduce the illumination on your slide and by lowering the condenser away from the stage. Wet mounts are often viewed with special types of microscopy to increase contrast and visibility, such as darkfield or phase-contrast microscopes (see module 3).

pure culture

of bacteria originates from a single cell. There are several ways to accomplish this task. The bacteria could be diluted in a sterile liquid, and then plated, or spread out over the surface of an agar plate. Robert Koch was the first to develop the streak isolation technique. The intention of the streak isolation method is to create an area where the specimen is so dilute that single cells do not overlap and can develop into a visible isolated colony. Spreading an inoculum which contains more than one organism will result in the isolation of the varied organisms. The inoculum used to prepare the streak plate could come from many different sources such as a clinical specimen (throat swab, blood or urine) , a swab from the environment or a pure broth, or solid culture. Seemingly impossible to determine, microbial biodiversity is truly astounding with an estimated one trillion coexisting species. Although particularly harsh climates, like the acidic environment of the human stomach or the subglacial lakes of Antarctica, may be dominated by a specific species, bacteria are typically found in mixed cultures. As each strain may influence the growth of another, the ability to separate and cultivate "pure" (consisting only of one type) colonies has become essential in clinical and academic settings alike. Pure cultures allow the identification and characterization of infectious agents from clinical samples. Whereas direct inoculation into broth carries the risk of generating a genetically diverse or even mixed bacterial population, plating and re-streaking creates a purer culture where each cell has a highly similar genetic makeup.

Non-motile

organisms appear to vibrate or shake caused by the collision of the cells with water molecules due to kinetic energy. This vibrational movement exhibited by non-motile organisms is known as Brownian motion. Remember, analyzing motility is another characteristic of an organism that may be useful in its identification.

Diplococcus

pair of cocci(2 cells)

Bacteria

prokaryotes

Agar plates

provide a larger surface area for culturing microbes than slants and may be employed to separate mixed cultures. We also use agar plates to identify cultural characteristics by studying colony morphology, as we saw in Module 02. We will use all of these types of media this semester.

negative stain

stains the background and leaves the bacteria unstained, improving the contrast between cells and background. When bacteria are stained using the negative staining technique, a dye solution with an acidic chromophore is employed. This type of dye carries a negative charge because it gives up a hydrogen ion, leaving it with a negative charge. The negative charge on the chromophore repels the negatively charged cell surface. The negative stain is particularly useful for determining the shape and arrangement of cells that are too fragile to be heat-fixed. In this quick and easy method, no heat fixing is necessary. In fact, the smear preparation is simply done by mixing a small amount of specimen with a drop of dye and then spreading this mixture across the slide using a second slide. Examples of commonly used negative stains are Nigrosin and India Ink. This is an example of a negative stain (above) using the acidic dye Nigrosin which repels the cells and stains the background a gray color. Note that cells are left unstained in this technique.

positive stain

stains the bacteria and leaves the background unstained. The charged region of a dye molecule that gives the dye its color is called a chromophore. Chromophores stain bacteria by forming ionic or covalent bonds between the bacterial cell and the dye. Because most cell surfaces are negatively charged, they are most easily stained by basic dyes with a positively charged chromophore. A positively charged dye with a basic chromophore gives up a hydroxide ion or picks up a hydrogen ion, giving it a positive charge. Examples of dyes that are often used for a positive stains include methylene blue or crystal violet.

Positive motility

test is indicated when the red pigment is observed radiating away from the central stab line (far right)