Microbio Lab Midterm

Aseptic Technique

procedures designed to prevent contamination by pathogens such as bacteria, viruses, fungi, and parasites

Culture Media

nutrient-rich preparations (liquid, solid, or semi-solid) designed to support the growth, isolation, and study of microorganisms or cells

Importance of heat fixing & air-drying specimens on slides

preserves cell morphology, prevent loss of specimens during staining, and ensure accurate microscopic diagnosis

Simple Staining

applying a basic dye to bacterial cells which are typically negatively charged.

Differential Staining

any staining technique that uses two or more dyes to distinguish between different types of cells

diplococci

pairs of cocci

Streptococci

chains of cocci

Staphylococci

grape-like clusters of cocci

tetrads

4 cocci in a square

sarcinae

cubic configuration of 8 cocci

coccobacilli

very short fat rods

vibrios

resemble rods, comma shaped

spirilla

rigid helices, will burrow

spirochetes

flexible helices

Gram-Positive Cells

stain purple, thick peptidoglycan layer, teichoic acid

Gram-Negative Cells

stain pink, lipopolysaccharide, outer membrane

Steps of a Gram Stain

food heat-fixed smear with crystal violet for 1 min, add iodine soln for 1 min, decolorize with alcohol briefly, counterstain with safranin for 1-2 min

Gram Stain - Primary Stain

crystal violet

Gram Stain - Counterstain

safranin (GS)

Gram Stain - Mordant

iodine

Gram Stain - Decolorizing Agent

alcohol

Purpose of a streak plate

used to separate microorganisms from a mixed population so that each colony arises from a single cell, creating a pure culture

How to do a streak plate

initial streak in one quadrant, then streak into second quadrant a couple of times from the first quadrant, then streak into third quadrant less times, then one streak into fourth quadrant

Pure Culture

contains only a single species of microorganism, typically obtained by isolating individual cells on sterile growth media so they multiply into genetically identical colonies

CFU

Colony Forming Unit, a microbiology measurement that estimates the number of viable bacteria or fungi capable of growing into visible colonies on nutrient media

Purpose of a Gelatin Stab, how to conduct test

test used to determine the ability of an organism to produce extracellular proteolytic enzymes (gelantinase) that liquefy gelatin

Positive reaction for gelatinase production

after incubating and then chilling, the medium remains liquid after chilling, meaning the gelatin has been enzymatically digested and will not re-solidify

Describe endospores, their function, and know which genera produce them

structures produced within certain bacterial cells that allow them to survive harsh conditions. Bacillus and Clostridium

Schaeffer-Fulton endospore staining Procedure

make a heat-fixed smear of Bacillus subtili, put a small piece of paper towel on top of the smear, saturate the paper towel with malachite green, place slide over beaker of boiling water and steam slide for five minutes. rinse with water, cover with safranin for 30 sec, rinse with water, blot slide with paper towel, observe

Endospore Staining - Primary Stain

malachite green

Endospore Staining - Counterstain

safranin (ES)

Why is heat used as a mordant in Endospore Staining?

keratin covering of endospores resists staining, so the primary stain has to be forced into the endospore through heat

What do endospores and their producers look like under the microscope after being stained?

Endospores stain green and cell stains pink

What bacteria are acid-fast? Characteristics that causes them to be acid-fast?

gram-positive cells with waxy mycolic acids in their cell walls

Kinyoun Acid-Fast Stain - Primary Stain

carbolfuchisn

Kinyoun Acid-Fast Stain - Decolorizer

acid-alcohol solution

Acid-Fast Stain - Counterstain

methylene blue

Appearance of Acid-Fast cells and Non-acid fast cells after staining

Acid-Fast cells stain red/pink and Non-acid fast cells stain blue

Bacterial Capsules and Functions

protective outer structure in bacteria and yeasts that is directly related to a microbe’s virulence

Negative Staining

staining the background around the cells - typically used for capsule staining

Method used to visualize capsules

Negative Staining - Dye stains background, but does not penetrate the capsules. Positive staining - dye stains the body of the cell. This leaves the capsule unstained (a halo)

Maneval’s A

stains the body of the cell - positive stain (acidic → red)

Maneval’s B

stains the background around the cell, not the capsule - negative stain (basic → blue)

Motility

the ability of a bacteria to move in their environment

Is E. coli motile

E. coli is motile

Is M. luteus motile

M. luteus is not motile

selective media

favor the growth of some microorganisms and inhibit the growth of others. selects for gram-negative bacteria

differential media

distinguish between different groups of microorganisms based on their biological characteristics (blood agar, MAC)

enriched media

general purpose media supplemented with highly nutritious substances such as blood (chocolate agar)

general purpose media (supportive)

support the growth of many microorganisms (TSA plate)

MacConkey Agar (MAC)

selective and differential. selective for Gram-negative bacteria to grow. Presence of lactose and neutral red (pH indicator) bacteria can use lactose as a carbon and energy source. Colonies of those that release acidic products are red.

Phenylethyl Alcohol Agar (PEA)

selective media. used to cultivate gram-positive bacteria

Blood Agar

enriched and differential. supports growth of many fastidious bacteria. differentiated based on their ability to produce hemolysins (proteins that lyse red blood cells). Hemolysis appears as a clear zone (beta/full) or greenish halo around the colonies (alpha/partial)

Bacterial Growth Curve

4 phases of bacterial growth shown over time

Typical phases of Growth for a population of bacteria

Lag, Exponential, Stationary, Death

Lag Phase

after inoculation of the cells into fresh medium. population remains temporarily unchanged. no apparent cell division occurring, cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc and increasing metabolic activity.

Exponential (log) Phase

growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission and are growing by geometric progression. cells divide at a constant rate depending upon the composition of the growth medium and the condition of incubation.

Stationary Phase

exponential growth doesn’t continue forever in a batch culture. growth is limited by one of three factors: (1) exhaustion of available nutrients, (2) accumulation of inhibitory metabolites or end products (3) exhaustion of space, in this case called a lack of “biological space.”

Death Phase

if incubation continues after population reaches stationary phase, this phase follows where the viable cell population declines. number of viable cells decreases exponentially

Carbohydrate (Glucose) Fermentation

used to detect the fermentation of specific carbohydrates. pH indicator. pH drops turning the broth yellow. Uses a Durham Tube

what is the purpose of the Durham tube?

traps gases. if bacteria produce gas as a by-product of fermentation, there will be an air bubble

Positive Carbohydrate Fermentation Test

the broth turns yellow (pH < 6.8)

Negative Carbohydrate Fermentation Test

the broth turns red (pH: 6.8-7.4)

Mixed Acid (Methyl Red)

used to determine whether a bacterium performs mixed acid fermentation of glucose and produces enough stable, strong acid end products to significantly lower the pH of its growth medium. Turns red in acidic environment

Positive Mixed Acid/Methyl Red Test

turns red in an acidic environment with the VP tube remaining unchanged

Voges-Proskauer

used to detect a precursor to butanediol. Turns red when positive.

Positive Voges-Proskauer Test

broth will turn red in the VP tube and remain unchanged in the MR tube

Positive Citrate Agar Test

green agar slant turns blue (alkaline pH) as citrate is used as a carbon source

Negative Citrate Agar Test

green agar slant remains green

Catalase Test

detects the presence of the catalase enzyme in bacteria by observing the breakdown of hydrogen peroxide into water and oxygen, indicated by bubbling

Positive Catalase Test

if bubbles are observed after adding Hydrogen Peroxide

Positive Oxidase Test

possesses cytochrome c oxidase indicated by purple TMPD

Starch Hydrolysis

detects whether a microorganism produces extracellular amylase, shown by a clear zone around growth on starch after adding IODINE

Reagent Used in Starch Hydrolysis

Gram’s Iodine (SH)

Positive Starch Hydrolysis Test

clear zone around growth.

Casein Hydrolysis

skim milk agar plate. used to determine whether a microorganism can produce the enzyme caseinase to hydrolyze the milk protein casein, indicated by a clear zone around bacterial growth

Positive Casein Hydrolysis Test

there is a clear zone around the growth, separating it from the untouched white agar

H2S Production/SIM

tests for 3 bacterial traits: sulfur reduction (H2S production), indole production from tryptophan, and motility in a semisolid medium. Uses Kovac’s reagent to detect indole.

Positive H2S production test

Medium turns black

Reagent used to detect indole in SIM tube

Kovac’s Reagent

Positive Test of indole production by Kovac’s Reagent

Red ring at the top of the medium

Urease Test

used to detect bacterial urease activity by identifying a color change (yellow to bright pink) caused by ammonia production when urease hydrolyzes urea

Products of Urea Hydrolysis

carbon dioxide and ammonia

If a bacterium produces urease what happens to the pH of the culture medium

the pH of the culture medium will increase

Indicator used for the urease test

Phenol Red (Urease Test)

Positive Urease Test

BRIGHT pink

Obligate Aerobes

organisms that has to have oxygen. clustered at the top

Obligate Anaerobes

organisms that don’t have to have oxygen. clustered at the bottom

Facultative Anaerobes

organism that can live in either oxygenated or non-oxygenated environments but prefer O2 (by default they are anaerobic). clustered towards the top, spread towards the bottom

Microaerophiles

like oxygen. small organisms. tube with the growth near the top of the tube. narrow window of O2 concentration

Aerotolerant Anaerobes

does not require oxygen for growth but can survive with it. spread out

Neutrophile

microorganisms that thrive in environments with a neutral pH (5.5-8.5)

Acidophile

microorganisms that thrive in environments with an acidic pH (1-5.5)

Alkaliphile

microorganisms that thrive in environments with a basic pH (7.5-11.5)

Water Activity

the amount of unbound water in a sample. measure of the amount of free, usable water available for microbial growth. 0 - no free water/dry. 1 - pure water

Halophile

microbes that require the presence of salt (NaCl) in their environment

Halotolerant

tolerance of high salt conditions beyond necessary for their growth