Biology Lab Practical Study Guide: Understanding Citrate Test, Hemolysis Types, and Aseptic Techniques

How can you differentiate between bacteria and filamentous mold on an agar plate? How can you differentiate between bacteria and yeast on an agar plate?

- You can differentiate between bacteria and filamentous mold on a plate because mold appears fuzzy.

- You can't differentiate between bacteria and yeasts on a plate because they look too similar. You must use a microscope to differentiate them.

What is the purpose of a quadrant streak?

The purpose of the streak is to obtain individual CFUs

What must you do between each quadrant for the streak to be preformed correctly?

You must sterilize the inoculation loop

What is aseptic technique?

Practices/procedures done to prevent contamination of pathogens

When transferring bacteria from a plate to a liquid broth, what must be done to the tube before closing the lid?

You must pass the lip of the tube over the flame to sterilize it

How do you know when your loop has been sterilized? Can you pick bacteria up immediately after sterilization, why or why not?

You know when the loop turns red. You can't pick up bacteria immediately after because it will be too hot and will kill the bacteria.

What is the total magnification of the 10x objective?

100x

What is heat fixation? Why must we heat fix a microbe to a slid before performing a stain?

- It's the process of mixing your specimen with DI water on a slide and using the bunsen burner to pass your slide over the flame until the water evaporates

- We must do this so the specimen adheres to the slide and doesn't wash off in the staining process

What is the function of the diaphragm on the microscope?

It works with the condenser to condense the amount of light going through the specimen

what is budding?

the asexual reproduction of yeast

what's the purpose of using a simple stain?

to be able to see the microbes more clear and as best as possible

when looking at a mold, what are the chains of spores called?

conidia

List the steps of the grams stain

1. Heat fixate

2. Add Crystal Violet (1min)

3. Rinse with DI water

4. Add Grams Iodine (1min)

5. Rinse with Decolorizer

6. Rinse with DI water

7. Add Safranin dye (1min)

8. Rinse with DI

what's the purpose of the crystal violet dye

it's to stain all cells purple

what's the purpose of the Gram's Iodine (mordant)

to lock crystal violet dye into the peptidoglycan to create a crystal violet-iodine complex

what's the purpose of the decolorizer?

it's to decolorize the gram-negative cells. It differentiates g-neg from gram-pos by making the gram-neg cells clear.

what's the purpose of the safranin dye?

this is what stains the g-neg cells pink so they can be differentiated from the purple g-pos cells.

what was the gram identify and cell shape of staphylococcus aureus?

gram-positive and cocci clusters

what was the gram identity of e.coli?

gram-negative single rods (bacilli)

Is a MAC plate selective, differential, or both? Describe why or why not?

It's both. It's selective for gram-negative organisms because it contains bile salts and crystal violet that inhibit the growth of g-pos. It's differential for lactose fermentation because it has a pH indicator, when pH drops below 6.8, there's a hot pink color change

Why do we need to see an old culture to see endospores?

when a culture gets old, that means nutrients are going to start to deplete, which is when the organism usually starts to produce endospores.

How would the cells appear if we forgot the decolorizer?

Cells would appear purple

What would cells look like if we forgot the Grams Iodine?

they would appear pink

You have an organism growing on an MAC plate. The media shows hot pink colonies. Interpret these results and give the name of the species that would show this kind of growth.

- Since there was growth that means the organism is g-neg.

- Since it turned hot pink, that means the organism is capable of lactose fermentation

Ex: E.coli

You have an organism growing on a MSA plate. The media is pink. Interpret these results and give the name of a species that would show this kind of growth.

- since there's growth that means the organism is salt tolerant

- since the media is pink that means the organism isn't capable of mannitol fermentation

Ex: S.epidermidis

why do we need to use heat for the endospore stain? Why do we need to use heat for the acid-fast stain?

- we need to use heat for the endospore stain in order to force the stain into the endospore since it has a hardy layer of keratin

- we need to use heat for acid-fast because of the waxy coating the cells have. The heat melts it, making it easier for the stain to go into the cells

is a blood agar plate selective, differential, or both?

It's differential to hemolysis (breakdown of RBC)

What are the types of hemolysis

-alpha : partial breakdown of RBC (greenish tint)

-beta: complete breakdown of RBC (clear)

-gamma: inability to breakdown RBC (red)

What's the difference between a plate being selective and a plate being differential?

- Selective means the organism selects for growth of a specific organism by inhibiting the growth of others

- Differential you'll be able to see a visual change. Media can be differential between an organisms ability to do something

Is a MSA plate selective, differential or both?

it's both. It's selective for salt-tolerant organisms because the media as a 7.5% salt content that inhibits the growth of non-salt tolerant organisms. It's differential to mannitol fermentation because it has a pH indicator and when it drops below 6.8, there is a yellow color change.

what types of organisms would you expect to see growing on a MAC plate?

Gram-negative organisms, and gram-negative organisms that can ferment lactose

You have an unknown organisms that you have inoculated onto a blood agar plate, after 48 hours you notice the media has good growth with a greenish tint. Describe the results and interpret them. Name a species that would show this result.

- This organism is alpha-hemolytic.

Ex: S.pneumoniae

You have an unknown organism that you have inoculated onto a blood agar plate, after 48 hours you notice the media has good growth with a clearing surrounding. Describe the results and interpret them.

- Beta-hemolytic

Ex: S.pyogenes

List the types of aerotolerance

1. obligate aerobe

2. obligate anaerobe

3. facultative anaerobe

4. microaerophile

5. aerotolerant

6. capnophile

what's an obligate aerobe?

requires O2 to survive (at top of tube)

what's an obligate anaerobe?

requires an-oxygenic environment (at bottom of tube)

what's a facultative anaerobe?

prefers 02 but can survive w/o it (middle of tube)

what's aerotolerant?

has no 02 preference (everywhere)

what's microaerophile?

requires below atmospheric levels of 02 (mostly at top but also throughout middle)

what's capnophile?

requires high concentrations of CO2

What's the media used for aerotolerance?

thioglycollate tube

what is aertolerance?

an organisms 02 requirements

you have an organism that is plated and grown on a MSA plate. There's good growth an media is yellow. Describe the results and give an example.

- Growth means the organism is salt tolerant

- Yellow means organism is capable of mannitol fermentation

Ex: S.aureus

you have an organism that is plated and grown on a MAC plate. There's good growth an media is yellow. Describe the results and give an example.

- Growth means gram-neg

- yellow means not capable of fermenting lactose

Ex: pseudomonas aeruginosa

what type of hemolysis do s.pyogenes and s.pneuominae show?

s.pyogenes: beta-hemolysis - complete breakdown

s.pneumoniae: alpha-hemolysis- partial breakdown

what's the difference between facultative anaerobe and obligate anaerobe?

facultative prefers 02 but can survive w/o it. Obligate anaerobe can't survive in 02

what types of aerotolerance do s.aureus, s.pyogenes, and clostridium sporogenes have?

- s.aureus: facultative anaerobe

- s.pyogenes: aerotolerant

- clostridium sporogenes: obligate anaerobe

what is the purpose of the catalase test? Are staphylococci catalase positive or negative. What about streptococci?

- purpose is to see if an organism has enzyme catalase present in order for it to break down the toxic form of o2 (h2o2)

- staph: positive

-strep: negative

what is the tube inside the phenol red broth called? What's the purpose?

Durham tube, it's purpose it to collect gas if it is produced by the organism

which test is used specifically to identify staphylococcus aureus?

coagulase

what is the purpose of the antibiotic susceptibility test?

it's to see if an organism is susceptible or resistant to a specific antibiotic

what's the purpose of the starch hydrolysis test?

it's to see if an organism has the exoenzymes present in order to breakdown start outside of the cell into smaller pieces for the organism to utilize it them

What's the purpose of the phenol red fermentation test?

to see if an organism can ferment a specific carbohydrate with or without gas production

I have inoculated a phenol red broth, after incubation I notice the broth is yellow with a bubble in the Durham tube. Describe the results

- the organism fermented a specific carb and it produced gas

what does IMVic stand for?

Indole, Methyl Red, Voges-Proskauer, Citrate

what's the purpose of indole test and how can you interpret results?

- to see if an organism can produce indole from tryptophan

- after adding Kovac's reagent, a red ring means positive

what's the purpose of methyl-red test and how can you interpret results?

- to see if an organism is capable of mixed acid fermentation

- after adding methyl-red reagent, a red color change is positive

what's the purpose of voges-proskauer test and how can you interpret results?

- to see if an organism can ferment glucose, specifically 2,3 butanediol production

- after adding VP A + B reagents, a red color change is positive

what's the purpose of citrate test and how can you interpret results?

- to see if an organism is capable of utilizing citrate as a carbon source

- growth alone means positive, or a blue color change

why do we need to add mineral oil to our decarboxylase broths?

we need it because it removes 02 and creates an anaerobic environment

why do we use immersion oil on 100x mag?

We use it because light will start to refract and make it hard to see the organisms.

I have inoculated a nitrate reduction broth, after the addition on nitrate reagents A+B, I notice a color change to red. What species of bacteria could show a result like this. Interpret the results

- this means the organism is positive for nitrate reduction from nitrate to nitrite and negative for denitrification

Ex: S.epidermidis

name a species of bacteria that has amylase enzymes and can utilize starch as a nutrient

bacillus subtilis

I have inoculated an MR-VP broth. After incubation, I split the MR_VP broth into two tubes. In one tube I have added methyl-reagent, the broth turned red. In the other rube I have added VP reagents A+B. After waiting one hour, the broth didn't turn red. Interpret the results of the two tests.

- The methyl-red tube is positive for mixed acid fermentation

- the VP tube is negative for glucose fermentation (2,3 butanediol production)

I have inoculated a SIM agar. After incubation, the media is black throughout. I add Kovac's reagent and a pink ring appears. Interpret the results.

- organism is motile

- organism is positive for sulfur reduction

- positive for indole production from tryptophan

what's the purpose of preforming a serial dilution on a urine sample?

to dilute the urine to get a countable plate in order to determine the severity of the infection and to determine the causing bacteria

the MR-VP broth starts as one medium. What do you have to do to the broth before adding reagents? Why is this step important?

- You have to split the broth into 2 separate tubes

- this is important because each requires different reagents and different amounts

why is it important to not only look for a color change to the citrate medium but also look for growth?

citrate is the only carbon source in the citrate medium

What would the results of e.coli be for a SIM test?

+ for indole

+ for motility

- for sulfur reduction

what's the difference between TSA and BA plate?

BA plate has 5% sheep blood

what does it mean for an organism to be ubiquitous?

it means the organism can be found everywhere, but not all species can be found everywhere. Specific species thrive in different environments

why do we invert our agar plates

to prevent condensation from the lid from dripping onto the plate and interfering with the growth of the organism

list 2 reasons we used perafilm

1. to keep lid and base together

2. to prevent moisture loss