BISC300 Lab Final

Deck of agar, pigments, staining, and so much more!

Actinomycete Isolation (AI) Agar

\-Lets Actomycte’s grow. Added sodium propionate is added to allow for anaerobic fermentation

Glycerol is added for the carbon source

MRS- De Man, Rogosa and Sharpe Agar

Selective for Lactic Acid Bacteria.

Inhibit growth of Gram-negative rods, microbial flora, and fungi.

MRS has peptone, which serves what purpose?

Pepton provides a source of carbon and nitrogen.

MRS what will Lactobacilli look like on the plate verses streptococci colonies

Lactobacillus species- flat, wide, and colorless

streptococci- smaller and cream color

Eosin Methylene Blue(EMB)

Selective for gram-negatives(inhibits gram+)

What causes the color change in EMB?

\-Acid produced in the lactose fermentation allows the eosin- methylene blue dye complex being taken up by bacteria and forming dark colonies

\-Differentiates between LF and NLF

What compounds in EMB cause the color change?

__Eosin Y & Methylene Blue__

What agar do *E. coli* turn green in?

EMB

Luria-Bertani(LB) agar/broth

\-nutrient rich medium used to cultivate bacteria.

\-most of the time it is used for antibiotic selection.

\*used in conjugation testing to ensure it occured

MacConkey Agar(MAC)

Selective for Gram negative. Inhibit Gram positive(bile salts and crystal violet).

Differentiates LF from NLF

What causes the color change in MAC agar?

__Neutral red__ indicator changes from colorless to red in presence when lactose fermentation occurs.

Mannitol salt agar(MSA)

Selective for *Staphylococcus* species. Does this fro the high amount of salt concentration

\-differentiates Staph aureus from other staph species.

What causes the color change in MSA.

__Phenol Red__ changes yellow when acid from fermentation of mannitol is produced

Motility Test Medium(MTM)

Agar deep shows motility- can be made with or without the indicator triphenyl tetrazolium chloride(TTC);red

Mueller-Hinton(MH) Agar

This is the standard media used for disk diffusion for antimicrobial susceptibility testing

starch absorbs toxic metabolites

Nutrient agar(NA)/ Nutrient broth(NB)

general purpose agar- supports growth of non-fastidious organisms

Phenol red carbohydrate test broths

detects the fermentation of various carbohydrates. The durham tube to the broth tube collects gass and shows the production of gas upon fermentation

What causes the color change for carbohydrate broth tests?

The __phenol red__ turns yellow in the presence of acid by-products

Pseudomonas F agar(PsF)

enhances the production of **fluorescein**(pyoverdine), a fluorescent, yellow-green pigment produced by some *Pseudomanas* species

Pseudomonas P agar(PsP)

enhances the production of **pyocyanine**, blue-green pigment produced by *Pseudomonas aeruginosa*

Sabouraud dextrose agar (SDA)

\-Used to isolate fungi and yeast species. USES an acidic pH of 5.6(the pH inhibits bacteria in mixed samples)

\-Dextrose acts as the carbon source. this also at high concentrations which osmotically stable fungi can tolerate, but bacteria can’t

what is the reason SDA is used for the cultivation, maintenance, and isolation of fungi and yeast species?

The acidic pH of 5.6 enhances the growth of fungi and acts as an inhibitor for bacteria

Sheep blood agar(SBA)

aka Blood agar plate(BAP)

Shows hemolysis but allows growth of many types of bacteria

Simmons citrate agar

Differentiates enteric bacteria based on ability to use citrate as carbon source.

What causes the blue color change of the simmons citrate agar?

When citrate is consumed, the pH rises, and the __bromothymol blue__ turns blue.

Triple sugar iron agar (TSIA)

Differentiates members of Enterobacteriaceae.

yellow butt= acid produced from fermentation of glucose

yellow above the butt(in slant)= bacteria can ferment either sucrose and/or lactose(if acid production continues)

H2S production(reduced form sodium thiosulfate) reaction with the ferric ammonium citrate forms a black precipitate.

When a TSIA has a yellow butt and red slant what sugar(s) is fermented?

only glucose

What causes the color change in TSIA?

__Phenol Red__ when fermentation occurs it creates a lowering in the pH that makes the agar change to yellow

Trypticase soy agar(TSA)

nutrient rich medium that facilitates the growth of a wide range of fastidious organisms

What is **prodigiosin?**

A pigment produced by Serratia marcescens which turns red at room temp

What color does **prodigiosin** look at 37C?

any other cream-colored gram-neg rod

Is **prodigiosin** water soluble?

It is a non water- soluble pigment.

In 95% Ethanol it turns peachy

In Chloroform it turns bright highlighter pink

Is **fluorescein** water soluble?

Yes it is water soluble

Is **pyocyanin** water soluble?

Yes it is water soluble

Staining based on cell walls- gram staining

gram positive- purple= very thick cell wall

gram negative- pink= very thin cell wall

Flagella stain shows what?

Monotrichous- single flagellum

Amphitrichous- flagella at both ends

Lophotrichous- tuff of flagella at one or both ends

Peritrichous- flagella surrounding the cell

Capsule stain

complex stain- stain the cell pink, then dye the background(with a stain that does not penetrate the capsule

What is negative staining?

its a physical process, not a chemical one in which a dye binds to cellular material

Endospore stain

gram stain will not work on spores leaving them clear

what is a simple stain?

the process of using a single dye

used to understand the size, shape, and arrangement of the organism

use dyes like __methylene blue__, or __crystal violet__

What is a complex stain?

a stain that involves multiple reagents

what is a differential stain?

a stain that divides bacteria into groups

Things that bacteria can favor at different values?

oxygen, temperature, nutrients, and light

mesophiles

grow at moderate temperatures

psychrophiles/thermophiles

reproduce in extreme cold

What is **staphyloxanthin**?

pigment that gives colonies a cream to a light yellow color

What is **sarcinaxanthin**?

pigment that gives colonies a gold to orange color

What is **canthaxanthin**?

the red-orange pigment produces pink colonies

What is **violacein**?

purple pigment

How do you calculate cfu/mL?

plate count\* Dilution Factor/ what was plated.

What is a selective media?

it is a media that contains components that promote the growth of select microbes

what is a differential media?

a media that can distinguish between organisms with unique characteristics due to differences in growth or appearances.

Give an example of a selective media

MSA- contain high concentrations of salt, making them selective for halotolerant or halophilic bacteria

Give an example of a differential media

SBA- allows growth of many different organisms but, differentiates them based on their ability to lyse or damage blood cells.

Give an example of a media that is both selective and differential

EMB-

differentiates E. coli from other gram-negative bacteria since it can take up the dye in the medium to make them green.

selects for gram-negatives and

differentiates between LF and NLF bacteria.

what is beta hemolysis

the area of clearing around the colony

what is alpha hemolysis

it creates greening- the agar turns a greenish brown

what can bacitracin distinguish

group A strep can be distinguished from other beta-hemolytic streptococci by its sensitivity to the antibiotic

What are coliforms

coliforms are bacteria that have origin in human feces, along with soil and surface water.

What do coliforms usually serve as?

coliforms are mostly not pathogenic so they serve as indicator organisms when testing water fro contamination.

(T/F) EMB and MAC are selective for gram negatives?

True

What are EMB and MAC both differentiate?

Lactose fermentors from non lactose fermentors

EMB- the cells look darker if they are LF and E.coli will be bright green

MAC- from colorless to red when lactose fermentation occurs

Explain what changes with EMB vs. MAC

With EMB the colonies themselves change color

while with MAC the agar is what changes color

What is the main purpose of TSIA?

It differentiates members of the *Enterobacteriaceae*

\-using fermentation of various sugars(Dextrose{glucose}, Lactose, and Sucrose)

What color is Simmon’s citrate at a neutral pH?

What happens when the pH is raised(more alkaline)?

The color is green at neutral pH however when the pH is alkaline it turns blue.

What happens during the urease test?

the ammonia produced during ureas breakdown caused the pH to rise and the broth turns pink(__phenol red__)

\*typically used for identifying *Proteus* species

What does IMViC stand for?

Idole, Methyl-red, Voges-Proskauer, in Citrate

During fermentation of lactose what is produced(in yogurt)

lactic acid- that is what gives yogurt its sour tast

What is horizontal gene transfer(HGT)?

lateral movement of genetic material between bacteria that are not direct offspring.

3 mechanisms for lateral gene transfer

1. conjugation
2. transformation
3. transduction

What is conjugation?

transfer of DNA through a pilus

What is transformation?

the uptake of DNA from the environment

what is transduction?

introduction of genes via bacteriophages

In conjugation what is the recipient strain and who is the donor?

recipient is the F- while donor is the F+

What is requirement for transformation?

bacteria must be competent

If. not naturally competent- we have to make them chemically competent. How is that done?

using calcium chloride- that allows for the uptake of the plasmid.

What is the Kirby-Bauer method(akd disk diffusion)

single concentrations of antimicrobial is put onto disks and the zoned of inhibition is measured for effectiveness.

What is a zone of inhibition?

clear zone on the agar, shows resistance vs. susceptibility of microorganisms to antimicrobials.

Define Bacteriostatic

drugs will inhibit the growth of bacteria but could allow bacteria to grow again in the substance is removed.

Define Bactericidal

drugs kill the bacteria, and thus can clear the infection

Define narrow spectrum drugs

effective only against members of one group of bacteria.

either Gram pos/ gram neg

Define broad spectrum drugs

effective against some members of both groups

Give an example of a bactericidal drug

\-penicillin

\-streptomycin

\-rifampin

Give an example of a bacteriostatic drug

tetracycline

nitrofurantoin(in most tissues)

Give an example of narrow spec drug

penicillin(gram positives)- targets cell wall synthesis

Polymyxin B(gram negatives)- disrupts cell membrane

Give an example of broad spec drug

streptomycin- inhibits protein synthesis

tetracycline- interferes with protein synthesis

rifampin- inhibits DNA-dependent RNA polymerase

nitrofurantoin- attack ribosomal proteins and interferes with protein synthesis

Name one drug that is bactericidal and narrow spectrum

Penicillin

Name one drug that is bacteriostatic and broad spectrum

tetracycline

When you have an object in focus under one objective, you can switch to the next objective and the object should still be mostly in focus. This is because the microscope is ______

parfocal

What can be used to clean objectives?

lense paper

Some bacteria have a sticky polysaccharide layer. What is this structure called?

Capsule

Total length of stage micrometer is

1mm

Most bacteria that are capable of movement use a special structure called___

flagella

Why do you heat fix the bacteria to the slide before staining?

adhere bacteria firmly to the slide

kill the bacteria without affecting the cell’s shape

The Gram stain differentiates bacteria into two groups based on

The amount of peptidoglycan in their cell wall

The gram stain is a differential stain, but why is it also considered a complex stain?

Because it requires more than one reagent

Pathogens are most often___ because they have adapted to grow best at body temp.

mesophiles

What are three conditions that can affect the growth of bacteria?

Oxygen, temp, nutrients & light

that can be favored at different value for different bacteria

You will be dying yeast using the simple stain in lab, however you won’t be using the Gram stain, because yeast are Eukaryotic and do not have___

Peptidoglycan

which of the following stains will you be doing in lab?

simple stain, gram stain