Microbiology Lab test #1

- study guide and chart

Describe key PPE and techniques (things that you do) for lab safety.

wear lab coats, tie back hair, wear gloves, wash hands, disinfectant surfaces, hold microscope with two hands

Helminths

• parasitic worms, viewed at low magnification

• body segments, worm head structure, worm eggs under microscope

Molds

• multicellular filaments that form a network

• observe details to branching patterns fo the filaments and spore structures

• pay close attention to the arrangements and morphology of spores, which are often key to the indentification of molds

streptobacilli

• chains of rods

3. Label parts of a microscope and be able to explain how it is used.

iris diaphragm- an adjustable aperture than controls the amount and angle of the light reaching the special, affecting contrast and resolution

4. What process is used to sterilize media for lab use?

• an inoculation loop is put into an incinerator

agar plates

are a solid culture medium that provides a large surface areas for the isolation of individual colonies and observation of colony morphology

6. What RODAC stand for? What is the purpose of this media?

An acronym for “Replicate Organism Detection and Counting”, a type of contact plate used for sampling microogranisms from flat surfaces

7. Do you label the front or back of a Petri plate? Why?

You label the back or bottom of a petri dish because if they lid were to fall off then you woudnt be able to identfiy whose it is, or what bacterium is on it

8. What is the purpose of an incinerator?

The incinerator steralize the inoculation loop by leaving the loop in it for a few seconds

9. What tool do you use to inoculate an agar slant versus an agar deep?

Inoculation needle- transfer small amounts of culture or to inocluate mediat that requires stabbing into a solid media, such as into an agar slant or agar deeps

inoculation loop- used to transfer larger amounts of culturem making them ideal for broth transfers and streaking on agar plates

10. Why are bacteria streaked for isolation?

• to obtain and individual, well separated sample

• see morphology

11. Describe the purpose and rationale of the “quadrant method”. How do you know if you

The purpose is to get individual colonies

The rationale is by spreading the bacterium out you can get them less dense and be able to see individual colony

12. List steps of the quadrant methods. Diagram how to streak bacteria for isolation.

Sterile loop is used to progressively dilute a samople across the surface of an agar plate

13. How do you tell if a culture is contaminated?

The agar plates has different morpholgy on the agar plate while there is only one bacterium on it.

14. Why do cells need to be heat-fixed to a slide?

To make the bacteria cells stick to the slide and stay put thorugh the staining and rinising steps.

15. How do you prepare a heat-fixed smear?

First you put small water drops on a slide, then inoculate inoculating loop with bacterium, they inoculate slide where the water is. Finally put on a heating plate and wait for the water to dry up.

16. Discuss the theory of the negative stain vs. positive stains.

Postive stain

• a staining method that uses basic dyes to directly stain microbial cells, causing the cells to appear colored against a lighter background

Negative stain

• a staining method that uses acidic dyes to stain the background rather than the micrbial cells, leaving the cells clear and visible against a dark background

19. Understand actions antimicrobial drugs / disinfectants

antibiotic: an antimicrobial compound that selectively inhibits the growth of or kills bacteria

Disinfectant: a chemical agent used on nonliving surfaces to reduce or eliminate microorganisms

20. Define MIC and be able to interpret MIC data table results

MIC- The lowest concentration of an antimicrobial agent that visibly inhibits microbial growth

21. Define ZOI and understand how to measure ZOIs

ZOI- The clear area surrounding an antimicrobial disc where microbial growth has been inhibited

you would measure the diameter of the ZOI in mm for your incubated bacteria

22. What is the purpose and rationale of the Kirby - Bauer disc diffusion test?

• A disc diffusion method used to evaluate bacterial susceptibility to antibiotics

• the rationale is antibiotics diffuse through agar and lyse susceptible bacteria

24. Understand the effects of UV light on a bacterial cell

The longer exposure to UV the less bacteria

25. Explain what capsules are and how they benefit pathogens

Capsule- protective outer layer composed primarily of carbohydrates that surrounds some bacterial cells

• benefit: being a shield against the host systems immune defense by its resistance to phagocytosis

26. Explain the rationale of the capsule stain

• Capsule is destroyed by heat fixing and washing away, so cant use simple staining

• so you color the background and the cell in order to see the halo

Escherichia coli: G+ or G-?

Gram negative

Pseudomonas aeruginosa: G+ or G-?

gram negative

Staphylococcus aureus: G+ or G-?

gram postive

Bacillus subtilis: G+ or G-?

gram postive

31. Describe two situations in which a Gram-positive cell may appear Gram-negative after conducting the Gram-stain.

1) the culture is old or the cell wall is damaged, so the thinned or degraded peptidoglycan cannot retain the crystal violet-iodine complex, or (2) the smear is over-decolorized with alcohol, which strips the dye from the otherwise thick peptidoglycan. In both cases the cell loses crystal violet and takes up the pink safranin counterstain

32. Describe one situation in which a Gram-Negative cell may appear Gram-Positive after conducting the gram stain

the smear is under-decolorized (the alcohol is applied too briefly or the smear is too thick). The outer membrane is not fully dissolved and the crystal violet-iodine complex is not washed out of the thin peptidoglycan, so the cell stays purple and fails to take up safranin

33. Be able to write out the 4 reagents for the gram stain and explain what is happening at each step

• Crystal violet stains everything purple.

• Iodine traps the purple dye inside the cell.

• Alcohol is the key step: Gram-positive cells keep the purple, Gram-negative cells lose it.

• Safranin turns the colorless Gram-negative cells pink.

Result: Gram-positive = purple, Gram-negative = pink.

Memory aid: Crystal violet → Iodine → Alcohol → Safranin = “Can I Add Salt?”

34. Be able to explain why flagella need to be stained with a special flagella stain

• they are so thin that the staining material needs to be built up

36. Be able to write out the reagents for the Acid-Fast Stain and what they do at each step.

• Carbol fuchsin stains everything red. Heat forces it through the waxy wall.

• Acid-alcohol is the key step. Acid-fast cells have mycolic acid in their walls, which holds the red dye tightly. Other cells lose it.

• Methylene blue turns the colorless cells blue.

Result: Acid-fast = red, Non-acid-fast = blue.

Memory aid: Red = acid-fast, Blue = not.

37. Be able to explain the importance of the Acid-Fast Stain

• identify tuberculosis and other mycobacterial infections

• identifies bacteria gram stains won’t

38. Give an example of an Acid-Fast bacterium.

Mycobacterium phlei

yeasts

• unicellular fungi are larger than most bacteria but smaller than molds

• usually oval or round

• look for features such as budding daughter cells and chains of elongated cells

Protist parasites

• usually in blood smears

• focus on elongated cell shape and the presence of a flagellum that runs the length of the cell that gives a “lacy” appearance

staphylococci

• clusters of circles

Agar slants

are a solid culture medium prepared in tubes to create a slanted surface, which provides some surface area for growth and is commonly used for maintiang cultures

Agar deeps

• solid culture medium prepared in tubes to create a small flat surface, which is useful for creating a low- oxygen environment at the bottom of the tube

Broths

• are liquid media that support microbial growth throughout the tube and are useful for obtaining high biomass of cells

Monotrichous:

one flagellum

Lophotrichous:

a lot of flagellum at one end

Atrichous

no flagellum

Amphitrichous

one at each end

cepalotrichous

multiple flagellum at both ends

periterichous

flagellum coming out of all sides

Phenol Red Broth: Purpose, Rationale, Methodology, Results/Interpretation

Purpose: Determine if bacteria can ferment specfic carb, producing acid/gas

Rationale: fermentation can produce gas or acid which lowers pH

Methodology: Flame-sterilize the loop and allow it to cool.

1. Inoculate by aseptically transferring a loopful of culture into the broth and mixing gently by swirling or rolling the tube. Do not shake vigorously, as this can introduce air into the Durham tube.

2. Incubate

3. Observe

Results: yellow = positive (fermentation)

Red = negative (no fermentation)

bubble= gas production

Urease Broth: Purpose, Rationale, Methodology, Results/Interpretation

Purpose: wether bacteria produce urease

Rationale: urea breaks down into ammonia, increasing pH

Methodology: Label each tube with the organism, date, and your initials.

1. Flame-sterilize the loop and let it cool.

2. Inoculate the broth with a heavy loopful of culture and mix gently. A heavy inoculum is important because urease activity is quantity dependent.

3. Incubate

4. Observe

Results: pink = positive for ammonium

tan= negative

Simmons Citrate Slant: Purpose, Rationale, Methodology, Results/Interpretation

Purpose: determine if use citrate as primary carbohydrate source

Rationale: citrate utilization produces alkaline conditions that change the indicator’s color

Methodology: Label the tube with the organism, date, and your initials.

1. Flame-sterilize the needle and let it cool.

2. Inoculate by streaking the surface of the slant in an S-shaped pattern.

3. Incubate

4. Observe

Results: blue= positive

growth= positive

green = negative

Starch Hydrolysis Test: Purpose, Rationale, Methodology, Results/Interpretation

Purpose: bacteria produce amylase to break down starch

Rationale: amaylase breaks down starch

Methodology: Label the bottom of the plate with the organism, date, and your initials. Divide the plate into sections if testing several organisms.

1. Flame-sterilize the loop and let it cool.

2. Inoculate by making a single streak line (or a spot, or a short zigzag) across the plate surface. Keep growth confined so the clear zone is easy to see.

3. Incubate

4. Flood the plate with Gram’s iodine and let it sit for about 30-60 seconds.

5. Pour off the excess iodine and observe right away, since the color fades.

Results: black/brown up to bacteria = negative

ring around bacteria= positive

******** change = negative********

SIMS Test

Purpose: hydrogen sulfide production, indole production, motility

Rationale: detects Hydrogen sulfide production, tryptophan break down, and bacteria movement

Methodology: Label the tube with the organism, date, and your initials.

1. Flame-sterilize the needle and let it cool.

2. Pick a small amount of growth from a single colony.

3. Stab straight down the center of the medium to about two-thirds of the depth (not to the bottom), then withdraw along the same line. Do not move the needle sideways, since that can mimic motility.

4. Incubate

5. Read motility and H₂S first, before adding any reagent.

6. Add Kovac’s reagent last: place 3-5 drops (about 0.2-0.5 mL) down the inside wall of the tube, without shaking.

7. Read indole within about 1-2 minutes, looking for a color ring at the top.

Results: black precipitate= hydrogen sulfide production

red ring = positive indole

Diffuse growth= motility

Kirby Bauer Disk Diffusion Test

Purpose: Find bacteria susceptibility to antibiotics

Rationale: antibiotics diffuse through agar and inhibit susceptible bacteria

Methodology: Prepare the inoculum

1. Select 3-5 well-isolated colonies of the same morphology from an overnight plate.

2. Suspend them in saline or broth.

2. Inoculate the plate “lawn”

3. Apply the disks

4. Incubate

5. Measure and interpret

1. Measure the diameter of the zone of complete inhibition (including the disk) to the nearest millimeter, using a ruler or caliper on the underside of the plate against a dark, non-reflective background (viewed with reflected light).

Results: Bigger ZOI = greater susceptibility to an antibiotic