Microbiome Techniques Lecture 2

Culture Media

used to grow microorganism to identify, study, transport, and store in the laboratory

culture media is ___ or ___ preparation

solid, liquid

culture media must contain ___ the nutrients required by the organism for growth

all

classifications of culture media

- chemical constituents from which they are made

- physical nature

- function

chemical composition media

based on exact knowledge of properties. defined (synthetic), complex

physical nature media

based on solidifying properties. liquid, semisolid, solid

function media

based on goal of the culture. supportive (general purpose), enriched, selective, differential

defined or synthetic media

all components and their concentrations are known

complex media

contain some ingredients of unknown composition and/ or concentration

example of something unknown in complex media

beef extract: extract of a cow, but how much protein, fat carbs, etc?

peptones (media component)

protein hydrolysates prepared by partial digestion of various protein sources

extracts (media component)

aqueous extracts, usually of beef or yeast

agar (media component)

sulfated polysaccharide used to solidify liquid media; most microorganisms cannot

degrade it. is derived from seaweed, similar to jello

functional types of media

supportive, enriched, selective, differential

supportive or general purpose media

support the growth of many microorganisms, ex: tryptic soy agar (e. coli, s. aureus, fungus, etc)

enriched media

general purpose media supplemented by blood or other special nutrients, ex: blood agar

selective media

favors growth of some microorganisms, kills growth of others, ex: MacConkey agar-selects for gram negative bacteria

___ ____ and ____ _____ in MacConkey agar inhibit the growth of gram-positive bacteria

bile salts, crystal violet

many terms are ___

interchangeable

differential media

distinguish between different groups of microorganisms based on their biological characteristics ex: blood agar (hemolytic vs nonhemolytic bacteria), ex: MacConkey agar (lactose ferments vs nonfermenters)

MacConkey Agar

lactose fermenters vs nonfermenters; lactose fermenting colonies produce acid, drop in pH changes color of agar

blood agar

hemolytic (RBC's are lysed, there is a halo around it) vs nonhemolytic bacteria (area around cells unchanged)

MacConkey agar uses ___ ______ as a selective agent. it's differential substrate (indicator) is ____ to differentiate between ____ and _____. major organisms inhibited are ____ ______

bile salts, lactose (red), fermenters (e. coli, klebsiella), nonfermenters (salmonella pseudomonas), most cocci

Mitis salivarius uses ___ and ____ ______ as a selective agent. it's differential substrate (indicator) is ____ to differentiate between ____ and _____. major organisms inhibited are ____ _____ and ____ _______

tellurite, crystal violet, sucrose (trypan blue), big > 2 mm (streptococcus salivarius--oral cavity), small < 1 mm (streptococcus mitis, other streptocci), staphyloccoci, enteric bacilli

Mannitol salt uses ___ ______ as a selective agent. it's differential substrate (indicator) is ____ to differentiate between ____ and _____. major organisms inhibited are ____ and ______ _____

7.5% NaCl, mannitol (phenol red), big/ yellow (s. aureus), small/ pink (staphyloccus epidermidis) streptococci, enteric bacilli

Sabouraud uses ___ ______ as a selective agent. It's purpose is to kill ___ and select ____ ______. major organisms inhibited are ___ ______

low pH (5.6) ± antibiotics, bacteria, cream colonies (fungi), most bacteria

isolation of pure cultures is...

population of cells arising from a single cell developed by robert koch

pure culture isolation allows for study of

a single type of microorganism in mixed culture

techniques used to isolate pure cultures

spread plate, streak plate, pour plate

streak plate involves technique of

spreading mixture of cells on an agar surface so individual cells are well separated from each other, involves use of bacteriological loop

in streak plates, each cell can

reproduce to form a separate colony (visible growth or cluster of microorganisms)

in spread plates,

a small volume of diluted mixture containing approx. 30-300 cells is transferred, spread evenly over surface with a sterile bent rod

in pour plates,

sample is serially dilated, diluted samples mixed with liquid agar, mixture is poured into sterile culture (some bacteria may grow on bottom of plate)

both pour plates and spread plates may be used to determine the number of

viable microorganisms in an original sample

Streak plates are ____ to determine the number of viable microorganisms in the sample. Why?

not, loop being dipped in original culture and being streaked but you do not know how much you picked up loop

serial dilution

original mixture diluted to factor of 10 multiple times, ideal range is finding colonies that when dilated, have 30-300 cells

tntc

too numerous to count--too many cells in plate can be difficult to count

how to determine colony-forming unit (CFU) of original sample?

plate count x dilution factor

measurement of microbial growth can measure changes in ____ and ____ of cells

number (human), mass (bacteria)

direct measurement of cell numbers can be done with

counting chambers

electronic counters-flow cytometry

on membrane filters

counting chambers

- easy, expensive, quick

- useful for counting both prokaryotes and eukaryotes

- cannot distinguish living from dead cells

direct count on membrane filters

• cells filtered through special membrane that provides dark background for observing cells

• cells are stained with fluorescent dyes

• useful for counting bacteria

• with certain dyes, can distinguish living from dead cells

Flow cytometry

• Microbial suspension forced through small orifice with a laser light beam

• As individual cells pass in a single file through a laser beam, the instrument

measures their physical characteristics (size and granularity) and specific fluorescent tags

viable counting methods

spread and pour plate techniques, membrane filter technique

how are viable counting method techniques counted?

after incubation number of organisms are determined by counting number of colonies multiplied by dilution factor, results expressed as colony forming units (CFU)

in membrane filter technique, bacteria from ____ samples are trapped on membranes of known pore size

aquatic

in membrane filter technique, membrane is ____ in culture media, colonies grow on ____, colony count determines number of bacteria in ____ ______

soaked, membrane, original sample

if microbe cannot be cultured on plate media, ____ are made and added to suitable media. _____ is determined to yield the most probable number (MPN)

dilutions, turbidity

measurement of cell mass can be determined by

dry weight, quantity of a particular cell constituent, turbidometric measures (light scattering, rate cloudiness of sample)

dry weight

time consuming and not very sensitive

quantity of a particular cell constituent

useful if amount of substance in each cell is constant (ex: protein, DNA, ATP, or chlorphyll

Turbidometric measures (light scattering)

quick, easy, and sensitive

turbidometric low absorbance

little/ no bacteria

turbidometric high absorbance

lot of bacteria

optical density (OD)

qualitative measure, not an exact number, related to turbidity

gram staining steps

1. crystal violet

2. gram iodine

3. alcohol

4. safranin (red dye)

crystal violet purpose

dyes cell same purple color

gram's iodine purpose

mordant (stabilizer), causes dye to form large complexes in peptidoglycan meshwork. thicker gram-positive cell walls are able to trap larger complexes

alcohol purpose in gram staining

dissolves lipids in outer membrane and removes dye from peptidoglycan layer of gram-negative cells

safranin purpose

gram-negative bacteria colorless, safranin acts as a counter-dye to indicate their presence

ways to distinguish microorganisms

• Colony characteristics

• Microscopic morphology

• Staining

• Growth conditions

• Biochemical tests (for definitive identification to species level)

how to determine if something is aerobic or anaerobic

pour agar into test tube, stab agar with loop. if it likes O2, it grows close to surface, if not, it grows away from surface

obligate aerobe

needs O2

obligate anaerobe

doesn't like O2

facultative anaerobe

could do with or without O2

microaerophile

likes some O2, but not a lot

capnophilic organism

wants CO2 instead of O2

what bacteria is an obligate anaerobe?

porphyromonas gingivalis, associated with periodontal disease, grows deep into tissue

enzyme profile tests

color change tests, specific enzyme test

color change test

if the bacteria secretes a certain enzyme, it will catalyze a substrate that will in turn result in color change (ex: use dye that changes color when pH changes)

specific enzyme test

set up assay to see if an enzyme can make microorganism go from a to b, ex: coagulase produced by s. aureus clots plasma

commercial kits for biochemical identification of bacteria

have reagents in tubes for anything you'd use to test for normally

• Spectrum of specific enzymes secreted by bacteria

• Ability to ferment sugars anaerobically

• Ability to assimilate sugars aerobically

• Usually 20 tests

Antibiotic susceptibility testing can be done with

• Qualitative (disc diffusion assays)

• Quantitative (determining the exact concentration of antibiotics needed to control the growth)

1.MIC (minimal inhibitory concentration)

2.MBC (minimal bactericidal concentration)

disk diffusion test

discs with different antibiotics placed on top of inoculated surface, bacterial sensitivity inhibits growth of test bacteria around disc, zone of inhibition (radius of bacteria free region) determines sensitivity extent, larger inhibition zone=greater sensitivity to particular antibiotic

Minimal inhibitory concentration (MIC)

different serial dilutions of antibiotics placed in growth medium, least concentration of drug that inhibits growth in tube that can be VISUALLY recorded

Minimum bactericidal concentration (MBC)

different serial dilutions of antibiotics placed in growth medium, least concentration of drug that KILLS ALL growth in tube that can be recorded

combining MIC with disc test

combine quantitative (MBC) with qualitative (disc/ MIC)