MCRO 251: Introductory Medical Microbiology - University of North Carolina at Chapel Hill

Aseptic technique

method used to minimize the contamination of pathogens

liquid media is also called

broth

solid media is also known as

agar

what does media contain?

Carbon, glucose, and other vitamins/nutrients required for microorganisms to survive

what are bacterial cultures?

in nature, bacterial cultures are a mix of several species living together in the same environment

to identify and study individual bacterial species, what has to happen?

bacterial cultures must be separated

Colony

offspring of a single cell

All cells in a colony are ______

genetically identical

why do we make streak plates?

to dilute bacterial cultures down to isolate single cells/visible colonies

Streak plate technique (4)?

1. Sterilize your loop before each step and after the final step

2. start by taking a small lapful of culture and gently streak it on one quadrant

3. streak across the previous quadrant into the neighboring quadrant 2-3 times, then streak the rest of the new quadrant without going back across

4. On the final streak, only go across the previous quadrant once before streaking the remaining empty agar

How do we estimate bacterial density?

We dilute liquid culture then plate the dilutions

When estimating bacterial density, ____ is too few colonies to count, _____ is too many to count

30, 300

Dilution factor?

1/dilution

colony forming units per milliliter equation?

(number of colonies counted x dilution factor)/ volume plated (in mL)

how should tubes be held when using aseptic technique?

at a slightly horizontal manner

why do we hold tubes horizontally?

to minimize the entry of dust, spores, etc. into tubes

when should inoculating loops be sterilized?

Before and after each use

what is the immediate objective of plate streaking?

to produce well-separated colonies from bacteria in mixed suspensions

when plate streaking, how should the loop be held?

slightly horizontal

what do you use to inoculate the butt of a slant?

needle

what do you use to inoculate the butt and the slant of a slant tube?

needle

what do you use to inoculate the slant only of a slant tube

inoculating loop

how should test tube caps be used/stored?

Always screw the cap and then loosen it by 1/4 of a turn to allow gas exchange

what is the greatest benefit of the plate count method?

it allows us to count only viable cells in the mixture

how do you determine CFU/ml of an original culture

add CFU/mL for all dilutions used (i.e. 10e-2 dilution + 10e-3 dilution)

gram-staining is a form of _____

differential staining

gram-positive staining

has a purple stain due to its thick peptidoglycan and lack of outer membrane

gram-negative staining

has a pinkish stain due to its thin peptidoglycan and presence of lipid membrane

5 steps of gram-staining

1. air dry and heat fix a slide

2. application of crystal violet

3. application of mordant (iodine)

4. application of 95% alcohol

5. application of Safranin (counterstain)

what is the purpose of crystal violet in gram stains

it is a basic dye that stains all bacteria purple

what is the purpose of the mordant in gram-staining

it reacts with crystal violet to make the cells hard to decolorize

what is the purpose of alcohol in gram staining

it is the differentiating step in which gram-positive bacteria will stay purple, and gram-negative bacteria will turn colorless

what is the purpose of Safranin in gram staining

it turns Gram-negative cells pink

what happens in under-decolorization?

G (-) cells will appear G(+)

what happens in over-decolorization?

G(+) cells will appear G(-)

function of the arm

supports the body tube is the part to grasp when carrying the microscope

function of the nosepiece

the rotating part to which the 4 objective lenses are attached

what are objectives?

lenses that magnify the images (low power is 4X, medium power is 10X, high-dry is 40X, and oil-immersion is 100X)

what objective lens is oil immersion?

100X only

Ocular lenses/eyepieces

lenses that magnify the image 10X

final magnification of a specimen is

ocular x objective

Body tube

connects the ocular lens with the nosepiece and objective lenses

mechanical stage

supports the microscope slide that is held onto the stage

Condenser

A lens located directly below the stage that concentrates the light before it passes through the specimen

Iris diaphragm

opening at the bottom of the condenser that regulates the amount of light entering the condenser

Course focus adjustment

rotating knob that moves the stage up or down to bring specimen into initial focus

fine focus adjustment

a smaller rotating knob within the coarse focus that brings the specimen into fine focus by moving the stage less distance

what organism use the capsule stain? what organisms use spore stains?

K. pneumoniae, Clostridiodes spp.

function of capsules

made up of polysaccharides and glycoproteins and protect bacteria from phagocytosis of immune cells

How does the capsule stain work?

The background is stained acidic, and the bacteria are counterstained with a basic dye; the capsules remain colorless and have a halo

what is the function of spores?

metabolically inactive spores protect bacteria from adverse environments

Spore Staining Procedure

1. endospore accepts the malachite green as the primary stain

2. vegetative bacteria use the safranin counterstain and look pinkish

what is selective media?

inhibits growth of certain bacterial species while also permitting the growth of others

what is differential media?

does not inhibit growth, but causes different species of bacteria to appear differently based on certain characteristics

what kind of media is mannitol salt agar?

selective and differential

how is mannitol salt agar selective?

it contains 7.5% of high salt concentrations to select for staphylococcus species since they are halo tolerant

how is mannitol salt agar differential?

it contains mannitol sugar and a pH indicator so mannitol fermenters turn yellow, while non-fermenters stay pink

what is MSA typically used for?

to differentiate S. epidermis (commensal species) from S. aureus

what kind of media is MacConkey agar

selective and differential

how is MAC selective?

it contains crystal violet and bile salts to select for gram-negative bacteria

how is MAC differential?

it contains lactose and pH indicator so that lactose fermenters turn bright pink, while non-fermenters stay yellow

What kind of media is sheep blood agar?

differential

Does sheep blood agar inhibit bacterial growth of some species?

No

B-hemolysis

complete rupture of RBCs and leaves a clear area around the colonies

Alpha-hemolysis

causes partial hemolysis of RBCs, but hemoglobin is oxidized by bacteria into methemoglobin to make a greenish color

gamma-hemolysis

contains no rupture of RBCs, and the plate stays red

what tests can be used to identify streptococcus species?

A disc/P disc and bile esculin slants

what are Lancefield groups

contains B-hemolytic streptococcus that are further grouped based on their carbohydrates in their cell walls

what organism is a group A streptococcus?

S. pyogenes

what organism is a group B streptococcus?

S. dysgalactiae

what streptococci are alpha-hemolytic?

S. pneuomiae and S. viridans

what does an A disc contain? P disc?

Bacitracin; optochin

what does the A disc differentiate between?

Group A strep vs. Non-group A strep species

In the A disc, what group is sensitive?

Group A (s. pyogenes)

In the A disc, what group is resistant?

Non group A (S. dysgalactiae)

In the A disc it is important not to include _____ because...

alpha-hemolytic strep because they are sensitive to bacitracin

What does a P disc differentiate between?

S. pneumoniae and other alpha-hemolytic strep

When the zone of inhibition is greater than 15 mm in a P disc test, the organism is _____

S. pneumoniae

what organism is sensitive to optochin? resistant?

S. pneumoniae; other alpha-hemolytic

what is the purpose of the candle jar?

Most streptococci are microaerophilic, so when the flame burns out, it reduces oxygen to 5-10%, giving the organisms the best environment to grow

what does the bile esculin slant differentiate between?

Lancefield group D (Enterococcus faecilis) and other gamma-hemolytic organisms

what organism can grow in Bile esculin slant? what cannot grow?

Enterococcus faecalis can grow and turn the tube black, while other gamma-hemolytic cannot

what does positive growth look like in the bile esculin slant?

it is black

purpose of Bile in the bile esculin slant

it is selective for organisms that can grow in the presence of bile

purpose of esculin in the bile esculin slant

it is a glycoside that bacteria can hydrolyze and consume for energy

what is the purpose of iron in the bile esculin test?

it reacts with components from esculin hydrolysis, turning the media black

how is alcohol the differentiating step of gram-staining?

Alcohol dissolves lipids and forms pores in gram-negative cell walls that cause them to leak macromolecules; Gram-positive do not have a lot of lipids, so leakage does not occur

Decolorization in old vs. young G(+) cultures

Old cultures decolorize faster than young ones

how do antibiotics affect G(+) cells in gram staining?

they appear G(-) because their cell walls have been damaged

what is the function of the oxidase test?

to differentiate Neisseria species, Pseudonomas, and Moraxella catarrhalis from other Enteric bacteria

in the oxidase test, which organisms are positive?

Neisseria spp., Pseudonomas, and M. catarrhalis

What does the oxidase test test for?

the production of cytochrome oxidase C

what is a positive oxidase test look like? negative?

dark blue color change; no color change

enzyme tests must use what?

colonies from solid media so the enzyme is present in sufficient amounts

what is important about the oxidase test?

it must be read within 10 seconds, because false positives that look pink occur

what is the function of the catalase test?

to differentiate between staphylococci and streptococci

explain how aerobic respiration and catalase go together (also H2O2)

In aerobic respiration, the final products are either water (cytochrome system) or hydrogen peroxide (flavoprotein oxidase system); Since H2O2 is toxic, the bacteria use catalase to break it into water and oxygen

In the catalase test, which organisms are positive? which are negative?

Staphylococci are positive; streptococci are negative

what is the function of the coagulase test?

to differentiate between pathogenic S. aureus and non-pathogenic S. aureus

what is coagulase?

It is produced by bacteria to convert fibrinogen to fibrin, which protects the cocci from phagocytosis from immune cells