Public Health Lab Test 1

Describe (or illustrate) how you would prepare a quadrant streak? Give explicit detail as to when you would sterilize your loop in particular.

Sterilize loop (hold it in flame, moving slowly so the entire length glows orange as it passes through the flame). Inoculate the loop in culture tube and streak it in zig zag in one quadrant of agar plate. Sterilize loop again, then pass it through half the the previous streak, drag the loop across the streak and into the adjacent quadrant, streaking again in zig zag. Sterilize the loop again the repeat the steps for third and then fourth quadrant.

Why would you need to do a quadrant streak (i.e. what is its purpose)?

To spread out so inoculum of culture on plate so that after incubation individual colonies will be present, which can later be used as pure culture.

What qualities can you use to describe a bacterial colony’s morphology? Include 4 traits and 2 examples of each.

Shape: circular, irregular, spindle, etc

Luster: shiny or dull

Texture: smooth or rough

Size: punctiform, small, moderate, large

What is an advantage and a disadvantage of using a spectrophotometer to measure the optical density of a broth culture? Describe the difference between what the spectrophotometer measures compared to what the plate count technique measures.

Advantage: You don’t have to wait for plating, incubating, then counting the plate and just hope that the plate is countable

Disadvantage: Less accurate at low cell concentrations

Measurements: Spectrophotometer measures visual turbidity while a plate count records the number of colonies that grew from dilution and notes them as colony forming units.

Without explicitly describing a specific staining technique, what is the purpose of differential staining?

A differential staining technique stains organisms based on some trait, creating a certain reaction if the organism is positive for that trait, and a different reaction (or no reaction) if it is negative for that trait.

Why is heat (supplied by steaming water) used in the endospore and acid-fast staining techniques?

Heat makes the endospores and the acid-fast cells more permeable to our stains of choice, malachite green and carbolfuchsin respectively, but not so much that the stain flows freely through the barriers. This allows the spores and cells to retain the stain as heat is taken away and some harsh decolorizer is applied to clear up the rest of the stain from the slide and vegetive cells and acid-fast-negative cells.

Ignoring wash steps with water, what are the steps of the gram stain technique?

First, you stain the heat-fixed cells with crystal violet for 1 minute. Then add Gram’s Iodine to the stain for 1 minute to fix the crystal violet in gram-positive cells. Follow that up with ethanol briefly to decolorize the gram-negative cells. Finally add safranin to the slide for 1 minute to counterstain any gram-negative bacteria.

How would the cells look if you skipped the first step?

You would end up seeing only gram-negative results (including false negatives), thanks to the crystal violet never being added.

How would the cells look if you skipped the second step?

You would see the same as in a., only see gram-negative results, but because the crystal violet was never complexed with iodine and was washed away by the ethanol.

How would the cells look if you skipped the third step?

Every cell would appear to be gram-positive, because the gram-negative cells were never decolorized, and so retained the crystal violet stain.

How would the cells look if you skipped the fourth step?

You would see gram-positive cells properly, but gram-negative cells would be decolorized and would appear clear.

For each of the following differential stains, what trait is being detected, and how does that quality cause the corresponding reaction in the context of the given staining technique? Gram stain

The gram stain detects the thicker peptidoglycan cell wall of gram-positive bacteria, which retains the crystal violet-iodine complex when subjected to a decolorization step, whereas gram-negative cell walls do not.

For each of the following differential stains, what trait is being detected, and how does that quality cause the corresponding reaction in the context of the given staining technique? Acid-fast stain

The acid-fast stain detects the presence of mycolic acid on the outside of gram-positive cell walls in some species. While this usually causes a muted gram reaction (weak gram-positive), this makes the cells also retain stain better once they have picked them up, which is facilitated by the application of heat. Thus, they retain the carbolfuchsin stain even when subjected to acid-alcohol decolorization.

For each of the following differential stains, what trait is being detected, and how does that quality cause the corresponding reaction in the context of the given staining technique? Endospore stain

The endospore stain detects the presence of spores generally, but the technique is primarily concerned with endospores, i.e. spores still within vegetative cells. The spores have tough keratin coatings that prevent easily staining them, but also cause them to resist decolorization by ethanol once they have picked up a stain. The staining process is facilitated by the application of heat.

For each of the following differential stains, what trait is being detected, and how does that quality cause the corresponding reaction in the context of the given staining technique? Capsule Stain

The capsule stain detects the presence of extracellular carbohydrate or peptidoglycan slime around cells. The stain makes the capsule apparent by applying a Congo red negative stain to the slide, which is repelled by cells and doesn’t stain the capsule, followed by the application of Maneval’s solution, which changes the Congo red to blue, and stains the cells blue, while not staining the capsule. This results in blue cells, a blue background, and a clear negative image of any capsules in the sample.

You’ve been given a culture that you’ve been told has a density of 5.4*10^8 CFUs/mL.

If you serially diluted the culture out some number of times and plated 0.1 mL (or 100 uL), how many (what number) colonies would you expect to see on the 5th plate in the series (from the –5 tube)? From the 6th plate? From the 7th plate? From the 8th plate? How would you classify each of these plates in terms of countability (too numerous to count, countable, too few to count)? (If you expect fewer than 1 colony, it is too few to count)

5th: Original* (.1mLx10^-5)=540 colonies too numerous

6th: 54 CFUs countable

7th: 5.4 CFUs countable

8th: 0.54 CFUs too few

Another member of your lab told you they diluted the culture to 1 in ten-million, plated 400 uL, and counted 23 colonies on the plate the following day. Do you think they actually used the same culture? What if they counted 54 colonies? (You can assume they performed the dilutions correctly and without contamination or other errors).

23 CFUs/ (.4 mL * 10^-7)= 5.75×10^8 CFUs/mL They used the same culture as 5.75 is not that different from 5.4

54CFUs/(0.4mL 10^-7)=1.3510^9 CFUs/mL This is not as similar to 5.4×10^8 so its safe to assume they did not use the same culture

The three domains of life:

Eukarya, Archaea, Bacteria.

Mention the steps in which you would sterilize the loop?

Pass the wire loop through a Bunsen burner making sure the wire and loop get red hot. Let the loop cool before use.

Why do you have to sterilize the loop?

To make sure it is sterile and you don’t contaminate your culture

What are the 2 forms that culture media come in?

Broths and Agar plates

What are 2 instruments used to isolate colonies

Inoculation loops and spreaders

List three of the seven categories that are used to describe colonies

Circular, Spindle, Irregular

In the following components of growth media, identify the source of carbon and the source of nitrogen (2 pts):

 Glucose

 Sodium Chloride

 Magnesium Sulfate

 Ammonium dihydrogen phosphate

 Dipotassium phosphate

 Distilled water

Glucose- source of carbon

Ammonium dihydrogen phosphate- source of nitrogen

The optical density of a culture is measured in a spectrophotomete T or F

True

What kind of environment do halophilic organisms prefer to grow?

High salt environments

Convert 150 μl to ml.

0.15 mL

Convert 1500 μl to ml.

1.5 mL

If you dilute out 0.1 mL of your cell culture, plating out 0.1 mL from each dilution, what is the CFU/mL if you count 37 cells from a tube that had been diluted out 1 to a million?

3.7×10^8 mL

If the domain Eukarya represents eukaryotic organisms, what are the two domains that represent prokaryotes?

Bacteria and Archaea

Identify one difference between simple and differential staining

Simple stains are general stains that stain all cells with same color, whereas differential stains distinguish cell types based on the final stain color.

What feature in bacteria belonging to the Mycobacterium genus allows for staining with acid-alcohol (ie., why are the cells “acid-fast”)?

Presence of the waxy lipid in the mycobacterial cell wall is the feature. And these waxy walls resist the acidic alcohol rinse and the cells remain stained.

The Gram-positive cell wall has thicker peptidoglycan than Gram- negative cell wall T or F

True

The Gram-negative cell wall has more lipid than the Gram-positive cell wall T or F

True

student is doing a Gram staining but forgets to do the decolorization step. What color are the Gram-positive and Gram-negative cells going to be? Justify your response.

They both are stained blue/purple. Without the decolorizing step the Gram-negative will retain the original crystal violet stain.

In the Endospore stain, how is malachite green forced into the endospores? Why?

Steam is used to force the malachite green. It is needed because the spore cell walls are resistant to permeation (or any similar/reasonable reasons why).

You are given four tubes each with 900 μl of diluent. You serially dilute your sample using 0.1 ml into the first tube, then 0.1 ml of that into the second and so on. From the last dilution, you plate 100 μl and the next day the plate has 200 colonies. What is the CFU/ml in each tube including the original bacterial culture?

200/.1mLx10^-4= 2×10^7 mL

Organize in the correct order the Gram staining procedure

1. Staining with crystal violet

2. Addition of iodine

3. Addition of the decolorizer

4. Staining with safranin

In a capsule staining procedure:

The background and cells are stained