micro lab midterm

What is the purpose of a lab coat

To protect your skin and avoid getting pathogens on your body

why is sheep blood agar differential?

distinguishes bacteria based on hemolysis of red blood cells

alpha hemolysis

partial red blood cell destruction- greenish brown zone

beta hemolysis

complete red blood cell destruction- clear zone around colony

gamma hemolysis

no red blood cell destruction – no change on agar

How is lens paper different from kimwipes?

Lens paper is soft, lint-free, and great for lenses because it prevents scratches

Kimwipes can cause scratches on lens glasses

Fine knobs are used for

Fine adjustment- stage moves slower

Coarse knobs are used for

larger adjustments- stage moves faster

What should be included on a label of your petri plate?

Name, date, ID # if known, media used

ocular X objective =

total magnification

Old petri plates and large disposal plastics should be disposed of im

Red biohazard in front of the room

Pure culture

culture made up of single bacterial species

What is the purpose of a streak plate?

spread out bacteria cells on the surface of the agar plate and obtain isolated colonies

Why do you invert (turn upside down) plates during incubation

To prevent condensation droplets from falling onto the agar surface and disrupting the growing colonies

Why do some bacteria require an acid-fast stain instead of a traditional gram-stain?

Acid fast = high concentration of mycolic acids, which repel aqueous stains used in the Gram staining procedure

Acid-fast bacteria

Retain the primary stain even when treated with acid alcohol. Cell walls contain mycolic acid.

Mycolic acid

Waxy, long-chain fatty acid in the cell wall of Mycobacterium that makes it resistant to acid-alcohol and contributes to acid-fast staining.

Mannitol Agar

Selective and differential medium; inhibits growth of organisms that are NOT halotolerant

Halotolerant

Able to survive and grow in high concentrations of salt.

Columbia CNA blood media

selective against (reduces) the growth of gram-negative organisms

Gram-positive will be _______ under microscope

Purple

Gram-negative will be _______ under microscope

Red/ pink

Why is handwashing important?

Removes microorganisms from your hands and helps prevent contamination and the spread of microbes.

Incubation

Keeping a culture under specific conditions (such as temperature) so microorganisms can grow.

Contaminants

Unwanted microorganisms that accidentally enter a culture or sample.

Colony

A visible mass of microorganisms growing on a solid surface, usually originating from one cell or group of cells.

Aseptic technique

method used to make the environment, the worker, and the patient as germ-free as possible

Sterilization

Complete removal or destruction of all forms of microbial life, including endospores.

Four general methods of sterilization

Heat, filtration, radiation, and chemicals.

Cleaning vs. sterilizing

Cleaning removes dirt and reduces microorganisms; sterilization destroys/removes all microbial life.

Axenic culture

culture containing only one species of microorganism and no contamination

How do you know a broth or slant is axenic?

growth should match the expected appearance

- check for signs of contamination, such as unexpected colony types or growth patterns.

Why should you mix a broth before use?

Microorganisms and nutrients can settle unevenly, so mixing distributes the cells throughout the broth and gives a more representative sample.

Inoculation

To introduce microorganisms into a growth medium so they can grow

Why is oil used with the 100X objective?

Oil reduces light refraction and allows more light to enter the objective, producing higher-resolution image.

Gram-positive bacteria

stain purple- thick peptidoglycan cell wall that retains the crystal violet-iodine complex.

Gram-negative bacteria

Stain pink/red- thin peptidoglycan layer and lose crystal violet during decolorization, then take up safranin.

Proper Gram stain: S. aureus color →

Positive- will be purple

If iodine is left out of a Gram stain, what happens to S. aureus?

will likely appear pink/red because iodine is needed to form the crystal violet-iodine complex that is retained by Gram-positive cells.

Proper Gram stain: E. coli color →

Negative- pink/red

If decolorizer is left out, what happens to E. coli? →

will appear purple because the crystal violet is not removed from the cells.

Gram-negative structure

thin layer of peptidoglycan. no teichoic acid. HAS outer cell membrane

When would you use a wet mount instead of a Gram stain?

When you want to observe living cells, especially their movement/motility, without staining or killing them.

Wet mount

a slide created using a coverslip, water, and a specimen

When would you use a simple stain instead of a Gram stain?

When you mainly want to see cell shape, size, arrangement, and general morphology

Why sterilize the loop between sections of a streak plate?

To dilute the bacteria across the plate and obtain isolated colonies.

Purpose of a streak plate

To separate individual bacterial cells and obtain isolated colonies from a mixed or concentrated sample.

Isolated colony

These are colonies separated from other colonies on a petri dish.

MacConkey agar

selective and differential medium - Gram-negative and lactose fermentator

Fecal coliforms

Gram-negative bacteria commonly found in the intestines/feces of humans and other warm-blooded animals.

MacConkey agar is selective for

Gram-negative bacteria.

MacConkey agar is selective against

Gram-positive bacteria

Lactose

Makes MacConkey agar differential by showing whether bacteria can ferment lactose.

Lactose fermenter on MacConkey

Produces acid and forms pink/red colonies.

Non-lactose fermenter on MacConkey

Does not produce acid from lactose and forms colorless/pale colonies.

Crystal violet + bile salts

Make MacConkey agar selective by inhibiting Gram-positive bacteria.

Purpose of PEA agar

Selects for Gram-positive bacteria by inhibiting many Gram-negative bacteria.

PEA agar: selective, differential, or both?

Selective.

MacConkey agar: selective, differential, or both?

Both

β-phenylethyl alcohol in PEA

Inhibits Gram-negative bacteria, allowing Gram-positive bacteria to grow.

Why inoculate Nutrient Agar from a PEA plate?

To transfer the bacteria that grew on PEA to a non-selective medium where they can grow normally and be observed more easily.

Mannitol Salt Agar (MSA) selective, differential, or both? →

Both

Role of sodium chloride in MSA →

The high salt concentration inhibits most bacteria but allows salt-tolerant bacteria such as Staphylococcus to grow.

Role of mannitol in MSA

allows differentiation based on whether bacteria can ferment mannitol.

Role of phenol red in MSA →

A pH indicator that turns yellow when acid is produced from mannitol fermentation.

MSA: yellow color →

Indicates mannitol fermentation and acid production

MSA: pink/red color →

Indicates no mannitol fermentation.

Purpose of Columbia CNA with 5% sheep blood agar →

Selects for Gram-positive bacteria/ differentiates for hemolysis

What makes Columbia CNA selective?

The antibiotics colistin and nalidixic acid inhibit many Gram-negative bacteria.

Columbia CNA with 5% sheep blood: selective, differential, or both?

both

Sheep blood agar: selective, differential, or both?

differential

What does sheep blood agar differentiate?

Bacteria based on their ability to cause hemolysis of red blood cells.

Rabbit plasma positive result →

Indicates that the organism produces coagulase, which causes plasma to clot.

Coagulase reaction →

Coagulase converts fibrinogen into fibrin, producing a clot.

What does pH measure?

The acidity or alkalinity of a solution

What can happen to organisms outside their normal pH range?

Their enzymes and cellular processes can be disrupted, reducing growth or causing cell death

Neutrophile

An organism that grows best at a near-neutral pH.

Acidophile

An organism that grows best in low pH

Alkaliphile

An organism that grows best in alkaline/basic conditions.

Halophile

"salt-loving" archaea that live in environments that have very high salt concentrations

-phile

prefers/requires the condition

-tolerant

can survive the condition but does not require it.

Mesophile

organism that grows best at moderate temperatures

Psychrophile

organism that grows best at cold temperatures.

Thermophile

heat loving

Obligate aerobe

Grows at the top of broth because it needs oxygen.

Obligate anaerobe

Grows at the bottom because oxygen is harmful to it.

Facultative anaerobe

Grows everywhere, but mostly at the top because it can use oxygen but doesn't need it

Why is the top of thioglycolate broth pink?

Oxygen is present at the top.

Resazurin

Indicator that turns pink when oxygen is present.

Endospore

A thick-walled protective spore that forms inside a bacterial cell and resists harsh conditions.

How long does it take a bacterial cell to develop an endospore?

Approximately 8-10 hours under favorable sporulation conditions.

Selective media

Media that allows certain microorganisms to grow while inhibiting others.

Differential media

Media that allows you to distinguish between microorganisms based on differences in their biochemical reactions.

What is the role of β-phenylethyl alcohol in the media? What does it do?

Inhibits Gram-negative bacteria, allowing Gram-positive bacteria to grow.

How are acid-fast cell structures different from other bacteria cells that are stainable by gram stain?

Thick, waxy, lipid-rich cell wall containing mycolic acid, which makes them resistant to Gram staining.

MSA is BOTH

Selective → high salt (NaCl) selects for salt-tolerant bacteria.

• Differential → mannitol fermentation changes the phenol red indicator

Sodium chloride creates (selective part)

high-salt environment that allows salt-tolerant (halotolerant) organisms to grow while inhibiting salt-sensitive organisms.

Phenol red is used to detect

the acid produced by changing color from red/pink → yellow.

Mannitol determines

whether the organism can ferment mannitol and produce acid.