Micro Lab Practicum

You have collected 4 plates after adequate incubation as shown below: 10-6: 465 colonies, 10-7: 280 colonies, 10-8: 20 colonies, and 10-9: no growth. Please answer the questions: Which plate did you select to calculate the CFU/ml? Why this plate was selected?

10-7: 280 colonies; 30-300 colonies/viable count;

Calculate the selected plate for counting (CFU/ml)

2.8x109CFU/ml;

This particular type of media, Mannitol Salt Agar, contains a selective ingredient that makes it inhospitable for most microbial growth. What is that ingredient (be specific)?

7.5% sodium chloride

Of the 2 SIM tubes shown here, which one is positive for Indole production?

A

Which of the following is not a part of the Gram stain?

A (Hydrochloric acid)

A positive test is produced when the substrate has been utilized and broken down into a neutral end product. What is the name of end product?

Acetylmethylcarbinol (AMC)

What causes the blue coloration change of the media?

Acidic conditions from hydrolysis

What is the purpose of staining samples?

B (to increase the contrast and visibility of the specimen)

Of the 3 TSI tubes, which ones show an Acidic Butt?

B and C

When should the lenses be cleaned? What is the correct way to clean them?

Before and after uses with lens paper and optical cleaning solution

How should agar plates be incubated? Why?

Bottom up. To prevent water condensation from the lid drops onto the culture causing contamination and to prevent the agar to dry out:

Which type of staining method would you use to determine endospore-forming cells from non-endospore-forming cells?

C/D (Specialized stain/Differential stain)

List the 3 lens systems on the light microscope.

Condenser lens, objective lens, and ocular lens:

What is the problem with the cultures in the tryptic soy agar (TSA) plates below? How to avoid it?

Contamination. Apply aseptic techniques

What is the function of the iris diaphragm?

Controls the intensity of light that passes through the stage and the specimen:

This is an Oxidase Dry Slide. What enzyme does a positive test reveal that the organism produces?

Cytochrome c oxidase

Which of the following is a characteristic of simple stains?

D (They are a basic stain AND can be rinsed with water)

What are the selective ingredients for this media?

Eosin and methylene blue

These structures can easily be rendered unviable by detergents, chemicals, and heat.

False

The black coloration indicates the production of what substance?

H2S or hydrogen sulfide

List two instances when the coarse adjustment knob is never used

High power objective lenses (40X and 100X) and while focusing the fine details of the specimen

In Tube A, what does the pink layer on top of the SIM media indicate?

Indole was produced

If you inoculated a sample onto an EMB plate and had the results of Plate A, what could you immediately conclude about that sample?

It is a lactose-fermenter It is a gram negative rod It could possibly be Escherichia coli All of the above

What reagent is added to the SIM media to visualize the above reaction?

Kovac's reagent

The name of the aqueous layer on the top of the SIM media is .

Kovacs reagent

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

Lift the lid of the agar plate to about a 45degree angle and work quickly close to the flame:

For what exoenzyme is this media testing for?

Lipase

Spread plate technique is most successful if the concentration of bacteria in the broth mixture is

Low

Looking at this Gram stain, the bacterial cells would be most likely to grow on MacConkey agar or Mannitol Salt Agar?

MacConkey or MacConkey agar

Specifically for the Methyl Red test, what reagent do we add (after incubation) to determine if the pH of the media has been changed?

Methyl Red Reagent

For the Vogues Proskauer, which media is split so that half is used as the VP test and the other half is used for the other test?

Methyl Red media

If you transfer material from an area of confluent growth on a streak plate to a tube of nutrient broth, can you be sure that the resulting broth culture is a pure culture? Why or why not?

No. Theoretically, an isolated colony of bacteria (CFU) refers to either a single cell or a clump of cells that descend from a single bacterial cell. In the area of a confluent growth on a streak plate, many microorganisms are deposited closely to each other and might merge/fuse together:

What type of sugar does the Urea Test utilize as a substrate?

None

Why is immersion oil used with the 100X objective?

Oil immersion prevents the refraction of light (focus the light rays) in order to increase the resolution (it has a refractive index identical to the glass that made the 100X objective lens)

In Tube B, what enzyme can we conclude that the organism produced?

Phenylalanine deaminase

________________ may be counted by diluting samples, plating the dilutions on solid medium, and counting the colonies that arise.

Plate count

How should a label be written?

Plate: bottom, test tube: on the side including organism name, name of person performed the work, date:

Provide three reasons why the use of aseptic technique is essential when handling microbial cultures in the lab or clinic.

Prevent contamination to the culture, yourself/other lab workers (no leaving contamination behind), and the public:

What changes the pH in Tube A?

Production of an alkaline end product

Name a common organism that would give a positive Urea Test.

Proteus

How can one tell that a decarboxylation reaction has occurred?

Purple coloration of media

What is the function of the field diaphragm?

Regulates the depth of the field of view

What genus of organism would likely produce the structure in the image?

Rhizopus

When using this agar, what is the sole carbon source available for the bacteria?

Sodium citrate

H2S is the byproduct of the breakdown of what ingredient in the TSI?

Sodium thiosulfate

What is the name of this agar?

Spirit Blue agar

What advantage(s) does the streak-plate method have over the spread-plate method?

Spread plate technique requires several steps: the original/mixed culture is diluted serially and a small volume of the final dilution is spread on the surface of an agar plate. If the inoculum is too concentrated, individual isolated colonies will not appear. Streak plate: the original culture, either in liquid or solid form, is directly diluted across an agar surface using an inoculating loop. This is a simple and rapid method. Pure cultures are usually obtained by the streak plate method:

Which square is positive for the production of the proposed enzyme?

Square B

How can you make sure that an isolated colony on a streak plate is really a pure culture?

Streak that isolated colony on a second plate (subculturing) for characterization:

How would this Simmons Citrate agar be inoculated correctly?

Superficial streak on the slanted surface

Define resolution.

The ability to differentiate two closely positioned objects via an optical instrument:

What would happen if, when storing the inoculated media in the incubator, the tube's cap was placed on too tightly?

The bacteria would die and no color change would occur.

Why do you need to use sterile inoculating loop or re-sterilize the inoculating loop between the 1st and the 2nd streaks and between the 2nd and 3rd streaks when doing a streak plate?

To dilute the bacterial cells/inoculum directly from the original sample/mixture of species across an agar surface:

The ultimate purpose of performing a serial dilution, plating the diluted bacterial samples on agar plates, and counting the viable colonies from the plate which contained 30-300 colonies is:

To find out the original concentration of the bacterial sample used for testing

Which gelatin hydrolysis tube illustrates a positive result?

Tube A

In regards to the Tubes shown in the image: Which one fermented glucose?

Tube A ferments glucose

Would the bacterial smear used for this gram stain be heat-fixed?

Yes

Scenario: You inoculated your bacterium into an ornithine decarboxylase tube and came back to the lab after 48 hours. You see absolutely NO change in the media from when you first inoculated it. What could possibly have gone wrong?

You forgot to place oil on top of the media before incubating.

A ____________ theoretically contains a single bacterial species

a)Pure culture

When 0.1 ml of the same dilution 10-4 is plated, the final plate dilution is:

a. 10-5

These structures help the bacteria to ________ other cells, tissues, and structures.

adhere

The gelatin is hydrolyzed into

amino acids

What is the name of the enzyme that bacteria may produce in order to break down Starch?

amylase

The order of growth seen in the first, second, third, and the fourth quadrant of a four quadrant streak plate:

b)Heavy confluent growth, dense growth, weak growth, and isolated colonies

The ____________ typically is used to separate microorganisms contained within a small sample volume, which is spread over the surface of an agar plate, resulting in the formation of discrete colonies distributed evenly across the agar surface when the appropriate concentration of cells is plated:

b)Spread plate

Name the structure that gives a halo appearance in this particular stain.

bacterial capsule

What does a positive result look like? (enter what is produced)

bubbles

One process by which yeast divide is though uneven cell division called

budding

The __________ procedure is designed to isolate pure cultures of bacteria, or colonies, from mixed populations by simple mechanical separation on an agar plate:

c)Streak plate

One yeast that can potentially cause several diseases is called

candida albians or candida

What is the name of the enzyme for which this reaction is testing?

catalase

Gelatin is a protein derived from

collagen

What is the purpose of the Durham tube inside this Carbohydrate Fermentation test?

collects gas

What are the stained structures represented in the micrograph here?

endospores or spores

In order for the reaction above to occur (yellowing of media), what do the bacteria have to do?

ferment the mannitol

The entire brush-like structure, containing the chains of ball structures is called the .

fruiting body

In the Starch media, the substrate is starch which is broken down into ______ subunits as end products.

glucose

In the test above, what is the name of the substrate that must be utilized for the outcome of the test to be positive?

glucose

Bacteria that can grow on this type of selective media are generally said to be

halotolerant

According to the growth on plate A and the results in the surrounding media, what has taken place?

hemolysis

What reagent is applied to bacteria to determine a positive or negative result?

hydrogen peroxide

In this media, there are 3 results to look for. By adding the reagent above, which result are you scoring?

indole

For this Starch plate, what reagent would need to be added in order to evaluate starch hydrolysis?

iodine

Looking at the growth on this MacConkey plate, we can conclude that this bacteria is a .

lactose fermenter, gram negative, or gram-negative rod

How is this media inoculated?

media is stabbed

The majority of the bacteria in this field of view have outer projections called flagella. What would these flagella be useful for?

motility or movement

In the Methyl Red media, how many acid end-products is the test detecting?

multiple

What is the ingredient in this media that causes the change of coloration due to production of acid wastes and lowering of pH?

neutral red

If you had a MacConkey plate that after incubation, had growth but no coloration, you could safely say that your bacteria is a

non-lactose fermenter or gram-negative

If the end products change the pH of the media, what ingredient (usually already added to the media) would allow us to determine that the pH has been changed?

pH indicator

In Tube A, the substrate has been broken down into its end products. What can we now conclude about the pH of the media?

pH is alkaline

This organism is most likely what Genus ?

penicillium

The substrate is broken down into 3 end products: ammonia, indole, and

pyruvate

What would a positive result look like when adding this reagent?

red

What is the name of this structure?

sporangium

The chains of ball-shaped structures in the image are referred to as .

spores or conidia

The structure stained here is not a reproductive structure. What primary function does this structure have?

survival

Why is it often necessary to dilute a sample before spreading it on a plate for a plate count?

to avoid confluent growth

What is the substrate that the bacteria must utilize for the positive reaction shown in the image?

tryptophan.

Question 1:This image shows what type of fungal structure?

zygospore