Microbiology Practical 1

• Correctly write the scientific name of a Gram negative and a Gram-positive bacterium

Staphylococcus aureus is positive (purple cocci) S. epidermis is positive (purple cocci) Pseudomonas aeruginosa is negative (pink or red rods)

Bacteria: concept of clones and colonies (e.g. from your air or ubiquity plate - What does each visible colony come from?). That is referring to the theoretical concept that - one microscopic bacterial cell over time develops into a macroscopic colony.

That is referring to the theoretical concept that - one microscopic bacterial cell over time develops into a macroscopic colony.

Proper use of the fine adjustment knob, for example, the order of the colored crossed threads viewed at 10X and 40X.

Yellow was on top

• Microscope - cleaning the lens system - how to.

at the beginning of each lab session, ensure the lens are clean with lens paper and blowing off dust at the end, be sure to wipe any oil immersion and clean objective lense with a lens tissue moistened with green soap and water

Know all the structures microorganisms use for motility

axial filaments, cilia, flagella, pili, and pseudopodia

prokaryotes: Cyanobacteria -Oscillatoria -Nostoc*

blue-green bacteria

Fungi: yeast - Saccharomyces morphology, size, reproduction (budding).

budding yeast

Primary stain

chemical dye, gives cells color

Decolorizing agent

chemical used to selectively remove a dye from one type of cell or cell structure

Prokaryotes: bacteria -3 types of bacteria

coccus, bacillus, spirochete

• What step is timing critical for proper staining, why?

decolorizing (alcohol/acetone); 5-15 seconds; can prevent proper staining

Pure Culture Technique: • Know how to recognize a contaminant in a streak plate.

growing in an area that you did not streak

Pure Culture Technique: • Know how to distinguish macroscopically a single isolated colony, why is it best for isolation of a pure culture?

it comes from a single cell

Be able to identify required prepared slides = the list written on the board

list below: look at pictures on phone from lab

• Bacteria: concept of clones and colonies - one cell produces a colony (as in Standard Plate Count Experiment).

one cell produces a colony (as in Standard Plate Count Experiment).

Fungi: -molds --Aspergillus sp. --Penicillium sp. --Rhizopus sp.

pictures in snapchat from lab

What would be the final color result of a stain if a step were left out or went too long?

red

• What would be the final color result of a stain if a step were left out or went too long?

remove all the primary dye from the primary step and render it a false Gram negative (pink and red)

Counterstain

stain applied to impart a contrasting color to cells that do not retain the primary stain

, Mordant

substance, technique used to bind, set, look dye in cell

Acid-fast stain: • Decolorizing agent, primary stains and counterstain.

Decolorizing agent (Acid alcohol), primary stains(carbol fuchsin) counterstain(methylene blue).

Pure Culture Technique: • What does a successful steak plate look like and how a streak plate is used to initiate a pure culture?

Dilution of bacterial cells divide and create a pure colony. Colony is a clone of cells that can be used for studying.

Pure Culture Technique: • Be able to distinguish macroscopically and microscopically (ie Gram Stain) among colonies of E. coli, S. epidermis, Serratia marcescens, and Micrococcus luteus.

E. Coli- S. epidermis- Serratia marcescens- Micrococcus luteus-

• Gram reaction, shape and arrangement of Escherichia coli and Staphylococcus epidermidis

Escherichia coli -gram reaction: negative (pink) -shape: rod -arrangement: clumps and singles Staphylococcus epidermidis -gram reaction: positive (purple) -shape: cocci -arrangement: clumps and few singles

Gram stain - Be able to distinguish Gram negative from Gram-positive bacteria.

Gram negative- pink -(due to counterstain safranin sticking to thin peptidoglycan cell wall) gram positive- purple -(crystal violet iodine dye sticking to thick peptidoglycan cell wall)

Acid-fast stain: • Why boiling water is used? What's the mordant?

Heat drives the carbol fuschin through the waxy lipid wall and into the cytoplasm

Know at least two ways to adjust light intensity on the microscope and the order you should adjust them

Iris diaphragm, Illumination Intensity Control

Know pitfalls of using the High-dry lens (40X). E.g. Do you use the coarse focus knob when the 40X is in position?

Just use the fine adjustment knob. The coarse adjustment knob should only be used with the lowest power objective lens.

Know the 4 objective lenses, magnifying powers, names, working distances, light required

Scanning (4x), Low Power (10X), High-power (40x), Oil Immersion (100x)

• Viewing Stained Smears and Describing the Shapes, Arrangements, Sizes (SAS) and Colors - Focus (10X and then oil immersion) on bacteria from 1 of your own slides or its equivalent, adjust light correctly, measure, draw and describe shape, arrangement, and staining characteristics etc.

Shapes -cocci (sphere) -bacilli (rod-shaped) -spirilla (spiral) Arrangements -coccus (single) -diplo (double) -staphylo (irregular, often grape-like clusters) -strepto (cocci arranged in chains) -sarcina (arranged cubes of 8) -tetrad (arranged in squares of 4)

Know the difference of Simple microscope vs Compound microscope and real image vs virtual image

Simple: only 1 set on lens, only enlarges the image, can be projected real image: what you see with the human eye Compound: 2 sets of lens (objective and ocular), cannot be projected, virtual image: reversed and backwards

• How is air contamination prevented when an inoculating loop is used to introduce or take a bacterial sample to/from an agar plate?

Since the opening of a plate is not readily flamed, one should hold the lid over the top of the open plate when inoculating so that air contamination is limited. Working near a flame is also useful.

Evaluation of Alcohol: • Why did we need a control (the left thumb)?

to determine the effectiveness of alcohol; to determine if any bacteria was removed from thumb onto the agar first press

Know how to use the dichotomous key to determine the identity of microorganisms observed in lab.

use the key by looking at the microorganism

Fungi (molds & yeasts) grow on Sabouraud's Dextrose Agar (SDA). Why do fungi grow better on SDA than TSA or NA (nutrient agar)?

SDA has a lower pH

Know what percentage of organisms are pathogens, helpful, beneficial or not harmful

99% good bacteria 1% bad

Define Working Distance, and what is its relationship to lens power (magnification)?

- As the power of the objective lens increases the working distance decreases

Recognize and name all microscope parts and their uses

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Microbiology tools used so far, what are they and their uses?

Petri Dish Agar Loop Microscope Bunsen Burner Tongs Striker

Evaluation of Alcohol: • Given the number of colonies in each quadrant, can you calculate the percent reduction?

% reduction= (colony 1st press) - (colony 2nd press) / (colony 1st press) X 100

Define Field of View and how its size (diameter) is related to magnification.

- The diameter of the field of an optical light microscope is the field number, which is the diameter of the field view in mm measured at the intermediate image plane. The field number is typically listed on the microscope eyepiece. Typically the lower the magnification of the eyepiece, the higher the FN is

Proper procedure when focusing the microscope and viewing a slide. (be able to write a step by step description and be able to demonstrate under timed exam conditions).

--coarse and fine adjustment knobs -Bring the object into focus by turning the fine adjustment focusing knob -Manipulate the diaphragm lever to reduce or increase the light intensity to get the clearest sharpest image -One Image is visual move the slide around to find what you're looking for -Position the slide on the stage with material to be studied on the upper surface of the slide -Turn on the light source using a minimum amount of voltage -Check condenser to make sure it has been raised to the highest point -Rotate objective so it is in line with the center of the stage -Turn the coarse adjustment knob to lower the objective until it stops

Know how to use the oil immersion lens (100X), its resolving power and how it reduces light scatter (see lab manual).

-A drop of oil should be placed on slide, the use of oil enhances the resolving power of the microscope, oil immersion reduces light diffraction and maximizes the numerical aperture to improve resolution

Demonstration area - various items and scopes at the side table especially those that are part of the microbial survey. Be able to recognize and describe

-Giardia lambdia; pathogenic protozoa (has monkey face) - Trichomonas vaginalis; pathogenic protozoa (has flagella) -Nostoc, cyanobacteria (filaments of moniliform cells in a gelatinous sheath

Define characteristics of Fungi, especially the molds and yeasts seen in lab.

-Heterotrophic (cannot synthesize its own food, dependent on complex organic substances for nutrition -Eukaryotic -Lack of tissue differentiation -Cell walls of Chitin or other polysaccharide propogate by spores -molds is a type of fungus that grows in multicellular filaments called hyphae. -yeast

Pure Culture Technique: • Know other methods described in the manual, text or class to achieve isolation besides the streak plate method and their advantages and disadvantages when compared to the streak plate method

-Pour plate method (Loop dilution): Liquefy three nutrient agar pours, cool to 50 degrees C, let stand for 10 min Shake mixed culture to disperse organisms, flame loop and mouths of tubes Transfer one loop of mixed culture to tube 1 Flame loop and mouth of tubes replace caps on tubes and return mixed culture to test tube rack disperse organisms in tube 1 by shaking Transfer one loopful from tube 1 to tube 2, flaming loop and mouths of tubes before and after transfer Shake tube 2 and transfer loopful from that tube to tube 3 flaming loop and mouths of tubes before and after transfer Pour each tube into a different plate to examine once dry advantage of this method = requires less skill than streak plate disadvantage of this method = requires more media, tubes, and plates

Culturing bacteria: tryptic soy agar (TSA) or nutrient agar (NA - not used so far), both general purpose media. SDA a special purpose medium. Blood agar and tryptic soy broth were also media that was used.

-TSA: all purpose -Sheep Blood Agar (SBA) : organisms that live inside of humans (in blood) prefer this agar -Sabouraud's agar (SDA): specific for fungi, pH is lower than TSA (more appropriate for fungal growth)

•Define resolution (resolving power). How do you maximize resolution? What is the relationship of resolution and magnifying power?

-The resolving power of a lens is its ability to completely separate two objects in a microscopic field -A Blue filter should be placed over light source, Condenser should be kept at the highest position, The diaphragm should not be stopped down too much, Immersion oil should be used between the slide and the 100X objective lens -In order for magnification to increase, resolution must also increase -best resolution: 1000X, Which lens: objective lens

Proper use of oil immersion and how to avoid problems associated with it use. E.g. How to avoid getting oil on the 40X?

-To use an oil immersion lens, first focus on the area of specimen to be observed with the high dry (40x) lens. Place a drop of immersion oil on the cover slip over that area, and very carefully swing the oil immersion lens into place. -Begin by focusing your sample using the 40x objective lens. Rotate the objective lens part way between the 40x and 100x lens so you can reach the cover slip on your slide.

Immersion oil - what is its function? Do you use the coarse focus knob when the 100X is in position?

-corrects refraction of light -no, The coarse adjustment knob should only be used with the lowest power objective lens.

Be familiar with Laboratory rules, regulations, and proper procedures. See Basic Microbiology Laboratory Safety. Be able to define the different Biosafety Levels and abbreviations (BSL)

-must wear closed toed shoes at all times -long hair should be tied back -gloves, contaminated paper towels, and disposable agar plates must be distinguished in biohazard bin biosafety level 1: not likely to post a disease risk BSL 2: poses a moderate risk to healthy adults, unlikely spread through community BSL 3: can cause disease, may spread, treatment available BSL 4: poses a lethal risk, no response to vaccines or treatment, deadly

Know the characteristics of Eukaryotes and Prokaryotes. Know all the characteristics for Protozoa, Algae, and Bacteria

-prokaryotes: No membrane-bound nucleus or organelles, single celled organisms, and are all bacteria or Archea -Eukaryotes: membrane bound nucleus or organelles, often multicellular organisms. Includes protozoans, fungi, algae, animals, plants. -protozoa -algae -bacteria

Simple Stain - be familiar with the definition; concepts of acidic and basic stains. Why they work (has to do with the charges on the cell walls we discussed in lecture). Is a simple stain a positive or negative stain? Is it acidic or basic?

-use of a single stain to color a bacterial cell -concepts of acidic and basic stains. Why they work (has to do with the charges on the cell walls we discussed in lecture). -positive -basic

Wet mount: how to make one correctly with no air bubbles. (be able to demonstrate and write a step by step description)

1 - Get a clean slide and cover slip. 2 - Place ONE small drop of water in the middle of the slide then and sample cells. Don't use too much or the water will run off the edge and make a mess! 3 - Angle the cover slip and place the edge on one side of the water drop. 4 - Slowly lower the cover slip (45 degree angle) on top of the water and cells. No bubbles 5 - Place the slide on the stage and view it first with the 4x red-banded objective. Once you see the image, you can rotate the nosepiece to view the slide with the different objectives.

• Provide three reasons why the use of aseptic technique is essential when handling microbial cultures in the laboratory.

1) does not contaminate the culture 2) does not contaminate the handler 3) no contaminates remain after

Capsule stain - How to perform the capsule stain. • Function of both the acid and basic dyes used [i.e. what do they stain (cell vs. background) & what are their colors?] • What the capsule looks like; and what is meant by the virulence factor. • Know an organism that has one. In lab, we assumed the organism was _____________.

1)Two loopfuls of the organism are used to prepare a thin smear 2) the smear is allowed only to air dry- do not heat fix 3)stain the smear with 1 % crystal violet for 2 minutes 4) the crystal violet is washed off the slider with copper sulfate into the proper waste container 5) Blot dry with bibulous paper -Crystal violet stains the cells and binds the milk proteins to the culture medium, causing the background to stain purple. Copper sulfate removes the violet and stains the capsules a light blue -bacteria. Other organisms with capsules are Bacillus anthracis, Streptococcus pneumoniae, & Klebsiella pneumoniae

Pure Culture Technique: • How to make a streak plate using aseptic technique. What is the objective of the streak plate technique?

1. Over a Bunsen burner, flame a loop until red, let cool 2. Uncap test tube, flame the top of the tube 3. Use loop to collect organisms by dipping to the bottom of the tube. 4. Reflame top of tube and recap 5. Remove lid to petri dish (holding over dish) Use the loop to streak one loopful of organisms back and forth over Area 1 starting near the rim of the dish 6. Flame the loop, cool, streak through area 1 one time and start making a back and forth pattern on Area 2. 7. Flame the loop, cool, streak through area 2 one time and start making a back and forth pattern on Area 3. 8. Flame the loop, cool, streak through area 3 one time, and start making a back and forth pattern on Area 4 9. Flame loop, and close petri dish lid. -isolate a pure culture from a mixed culture source

Know the Eyepieces (ocular lenses), magnifying power of these lenses

10x

Acid-fast stain: • Color of acid-fast bacterium, color of nonacid-fast materials (those stained with the counterstain) on slide.

Acid Fast- Pink Non-acid- blue

Spore stain: Example of a spore-forming bacterium Bacillus subtilis or B. megaterium. Others were mentioned in lecture.

Bacillus Clostridium

Be able to distinguish macroscopically (macro-inspection) the following colony types in a Petri dish: mold, yeast, bacteria; counts; growth or no growth

Bacteria: small and smooth Yeasts: medium and gooey Molds: large and fuzzy

Spore stain: Why is boiling water used? What's the mordant for this staining procedure?

Because the spore is heat resistant and needs high temperatures to force malachite green in endospores heat is the mordant

Pure Culture Technique: • Although 3 bacteria were in the original mix, all 3 seldom showed up in the finished product/plate. Suggest why not.

Competition among the different bacteria Dilution

Light effect on contrast and how to adjust the iris diaphragm lever to get the best image

Manipulate the lever to reduce or increase light intensity to create the clearest, sharpest image, in order to improve the contrast.

Acid-fast stain: • Example of acid-fast organism (Mycobacterium tuberculosis, M. smegmatis, or M. leprae).

Mycobacterium tuberculosis Mycobacterium smegmatis Mycobacterium leprae). Nocardia

Acid-fast stain: • Which acid-fast organisms are pathogenic and which are not.

Mycobacterium tuberculosis are pathogenic Mycobacterium leprae are pathogenic Mycobacterium smegmatis is non-pathogenic , Staphylococcus epidermidis is not pathogenic

Aseptic technique: • How to handle cultures (plates, tubes with agar and broths) to prevent contamination of you and other cultures.

Plates: cover is raised and held diagonally over the plate to protect surface from any contamination in the air. Tubes Agar/Broth: cap is removed, mouth is flamed, cap kept in hand, after culture is inoculated, mouth is reflamed & tube recapped.

• Primary stain, mordant, decolorizing agent, and counterstain.

Primary stain (crystal violet) mordant (iodine) decolorizing agent (acetone/alcohol) counterstain (safranin)

Spore stain -Reagents used (Primary stain = malachite green, Decolorizing agent = water, Counterstain = safranin) and the function of each (what do they stain - colors?)

Primary stain = malachite green (stains endospores) Decolorizing agent = water (decolorize vegetative cells) Counterstain = safranin (stain the destained vegetative cell )

How to put away a microscope properly including the placement of the cord and low power objective

Remove slide from stage -If immersion oil has been used, wipe it off the lens and stage with a lens tissue -Rotate the low-power objective into position -If the microscope was inclining return it to an erect positon -If the microscope has a built in movable lamp raise the lamp to the highest positon -Wrap the electric cord around the base -Adjust the mechanical stage so it does not project too far on either side -Replace the dustcover -Return Microscope to its correct place

Negative stain - done as a demonstration primarily for the purpose of its relevance to a capsule stain (next). Know examples and how the negative stain differs from positive stains like those used in the simple or Gram stain.

They are acidic and negatively charged and stain the background (India ink or Nigrosin) done as a demonstration primarily for the purpose of its relevance to a capsule stain -Crystal Violet and Safranin are positively charged and stain the cell usually cells appear as transparent objects against a dark background

What does a parfocal lens system mean? Define parfocal

This means that the image will remain in focus when changing from a lower-power objective to a higher-power lens.

Determine total magnification (objectives lens power X ocular lens power).

To get the total magnification take the power of the objective (4X, 10X, 40x) and multiply by the power of the eyepiece, usually 10X. -ocular lens x objective lens = total magnification

Spore stain: Colors of vegetative cells and spores after staining

Vegetative - Red Endospore - green

Pure Culture Technique: • - be able to distinguish colors of macroscopic colonies such as:

white - E. coli red - Serratia marcescens yellow - Micrococcus luteus.

Evaluation of Alcohol: • Was there a difference between swabbing the thumb and dipping the thumb?

yes, dipping for 10 seconds was not as effective as swabbing in removal of bacteria