Micro Lab Midterm

Two Tube Transfer

1. smack that thing

2. pick up and place tubes in hand

3. sterilize loop

4. uncap tubes

5. sterilize tubes- 3 or 4 s

6. transfer culture

7. recap tubes

8. sterilize loop

9. place everything back

cationic dyes

positive ion that exhibits color

methylene blue or crystal violet

anionic dyes

negative ion that exhibits color, doesnt penetrate cell, no heat fixation

acid fuchsin, congo red, or nigrosin

how long should heat fixation be for

10 seconds

resolving power of our microscopes vs average

around 0.2 µm vs 1.0

resolution equation

d = λ / 2NA

NA is numerical aperture or light gathering capacity of the lens

example of vibrio

V. cholerae

example of spirochete

Spirillum volutans

example of club shaped

Corynebacterium diptheriae

what type of stain is a gram stain

differential

history of gram stain

developed by danish Hans Christian Gram in mid 1880s

tried to differentiate bacteria causing pneumonia from lungs of deceased patients

considered stain a failure

pour plate

culture diluted in molten agar, poured into empty sterile petri dish and solidified

primary and secondary streak

primary: isolation of mixed colonies from each other on first plate

secondary: isolation of a single colony from first plate

general purpose media

maintain culture

nutrient agar (NA), tryptic soy agar (TSA), brain heart infusion agar- enriched (BHI)

selective media

inhibitors prevent growth of certain organisms

phenyl ethyl agar (PEA), eosin methylene blue (EMB), MacConkey agar (Mac)

differential media

growth of many microbes differentiated w/ indicators like dyes (fermentation causes pH change and color diff)

blood agar (BA), eosin methylene blue (EMB), MacConkey agar (Mac)

combination

selective and differential

Mac and EMB

complex media

undefined chemical composition, nutrients are extracts or digests from naturals

BHI and TSA

PEA

phenyl ethyl agar

selective only, inhibitor: phenylethyl alcohol, prevents gram neg cells

BA

blood agar

differential only, indicator: rbc, ex. TSA w/ 5% sheep’s blood, detects hemolysis, Beta- Best, Alpha- Average, Gamma- Garbage

cannot detect gram reaction

Mac

MacConkey agar

combination, indicator: neutral red for lactose and non-lactose fermenters, inhibitor: crystal violet and bile salts, block gram pos, used to show gram neg enterics (Enterobacteriaceae)

EMB

eosin methylene blue

combination, inhibitor and indicator: eosin and methylene blue, lactose and non-lactose fermenters, screens for coliforms (fecal, also UTIs)

2,3-butanediol in EMB

pale pink lavender color

used by Enterobacter aerogenes

mixed acid in EMB

produce very dark, black, green metallic sheen

motility agar

detect mobile flagella, soft agar, dye is optional (tetrazolium chloride) and turbidity or cloudiness is used, single line slab- motile spread away from line

antimicrobial agents 3 kinds

• chemicals that kill or inhibit growth of microbes

• disinfectants: strong, inanimate surfaces, not as effective as sterilization, may not kill spores

• antiseptics: safely applied to living tissue, prevent infection

• antibiotics: systemic circulation, most are synthetic analogs of naturals

disk diffusion asssay

• tests effectiveness of antimicrobial compounds

• E. coli and S. aureus

• sterilize foreceps, dip in alcohol and ignite over burner, always downward facing, pick up disks and dip in betadine, hydrogen peroxide, or lysol bleach, transfer to plates

kirby bauer test

• highly standardized testing antibiotic sensitivity

• lawn inoculation, stamped with antibiotics and incubated on MH

• zone of inhibition measured in mm and compared to table

• mueller hinton (MH) agar or broth

MH agar or broth

mueller hinton

used in kirby bauer test, simple nutrient composition, neutral pH (7.2-4), 4mm depth for lateral antibiotic diffusion

too low of bacteria inoculated kirby bauer

overestimation of sensitivity to the antibiotics

too high of bacteria inoculted kirby bauer

swamp antimicrobial or deplete nutrients

agar plates that test for exoenzymes

lipase

milk agar

starch agar

tube media that tests for metabolic processes

litmus milk

sugar fermentation broth

Kligler’s Iron agar

nitrate broth

gelatin

urea broth

phenylalanine

SIM

IMViC

litmus milk

• contains: lactose, casein and other peptones, litmus pH indicator

• test for: fermentation lactose, metabolism proteins, degradation casein, litmus reduction

• results in: acid production (pink), alkaline production, (blue) curd, redox (white is reduced), proteolysis (clearing)

in litmus milk, what does deamination of peptones produce

a blue, alkaline production usually at the top of the tube due to oxygen dependence

will litmus milk function as a pH indicator if it is in a reduced state

no, only when oxidized, so it will be white if reduced

what results in proteolysis for litmus milk test

caseinase enzyme to degrade casein

Phenyl Red (PR) Sugar fermentation broth

• contains one sugar: glucose, lactose, or mannitol

• indicators: phenyl red and a durham tube

• fermentation: phenol red turns yellow, gas bubble in tube

• can’t ferment: phenol turns pink (cerise), peptone degradation releases ammonia

Kligler’s Iron Agar (KIA)

• tests for: lactose, glucose fermentation, and sulfur reduction

• ingredients: 1% lactose, 0.1% glucose, 1% peptone (cysteine- ammonia sulfur)

• indicators: phenol red, iron from ferric ammonium citrate (combines w/ H2S to form black precipitate)

• lactose fermenters: 1% lactose + 0.1% glucose > 1% peptone, whole tube yellow

• glucose only: 0.1% glucose < 1% peptones, need O to breakdown so red on top, bottom is yellow

• reversion: oxidation of weaker less stable acids at top of tube, Enterobacter aerogenes

• sulfur reduction: H2S combines w/ iron, black precipitate at bottom of tube

lipase plate

• differential, tests for enzyme lipase

• lipase: hydrolyzes fats into glycerol and fatty acids

• indicator: spirit blue

• hydrolysis produces dark blue zone around growth w/ no oily surface

milk agar plate

• differential, tests for enzyme caseinase

• caseinase: hydrolyzes casein (milk protein) into amino acid products

• clearing is a positive result

starch agar

• differential, tests for enzyme amylase

• amylase: breaks down starch into simple sugars

• indicator: iodine added after colony growth

• iodine and starch make a darker purple ring, and a clearing

biochemical tests

catalase

oxidase

fermentation v respiration similarities

both see conversion of molecules into ATP, both see glycolysis (glucose into pyruvate)

respiration

• final e- acceptor O: aerobic

• final e- acceptor Nitrate, Sulfate, etc: anaerobic

• glycolysis → kreb’s cycle → ETC

ETC

transfer e- to molecules that have a more positive reduction potential, indirectly testing for presence of ETC

flavoprotein → FeS cluster → ubiquinone → cytochrome C → cytochrome C oxidase → final e- acceptor

catalase test

• aerobic metabolism produces ROS: superoxide radical, H2O2

• aerobic and facultative aerobic break down ROS w/ SOD and catalase

• 2 H2O2 → 2 H2O + O2

• add 3% H2O2 to edge of colony, kills organism, bubbles means catalase is present

cytochrome C oxidase test

• cytochrome C oxidase: removes e- from cytochrome C (oxidizes) and transfers to O (reduces)

• chromogenic reducing agent: dimethyl-p-phenylenediamine, aromatci amine

• ^^ when oxidized by cytochrome C oxidase, changes color to purple blue and passes e- back to cytochrome C

nitrate respiration test

• nitrate (NO3) used as final inorganic e- acceptor

• needs enzyme nitrate reductase to convert nitrate to nitrite

• or another to reduced nitrate to N2 (g) (denitrification)

• or nitrate to ammonium for incorporation (assimilatory nitrate reduction)

nitrate broth

• detects nitrate reductase (nitrate to nitrite, NO3 → NO2)

• reagents: nitrate 1 (sulfanilic acid) and nitrate 2 (dimethyl-alpha-naphthylamine)

• ^^ reacts with nitrite to form brick red color

• zinc powder can be used as a catalyst for rxn if color doesnt change to confirm that microbe is negative for nitrate reductase

• ^^ if tube remains turbid clear it has been reduced to N2 (g)

urea broth

• detects urease: degrades urea → 2 ammonia (NH3) and CO2

• pH increases to more alkaline, changes color to cerise (hot pink)

• indicator: phenol red

• pH > 8.1 (cerise); neutral (red); decreases (yellow)

gelatin

• detects presence of gelatinase: catalyzes hydrolysis gelatin → aa

• viewed after chilling in ice bath

• liquid is positive result

phenylalanine slant

• detects production of enzyme phenylalanine deaminase

• breaks phenylalanine → phenylpyruvic acid (PPA) + ammonia (NH3)

• reagent: 5-10 drops 10% ferric chloride (FeCl3)

• positive reaction changes yellow to green (pp in pool turns green)

SIM

3 results in single tube

• S: Sulfide; bacteria produces H2S and it reacts w/ Fe → FeS; black precipitate

• I: Indole; breakdown of tryptophan by tryptophanase produces this; 3-5 drops Kovac’s reagent; positive produces pink color

• M: Motility; determined by turbidity

fermentation

• final e- acceptor is organic molecule

• does not produce as much energy as respiration

• e- onto pyruvate → general end products organic acids and gases

  • mixed acid fermentation

  • 2,3-butanediol fermentation

fermentation general term

breakdown of polysaccharides into monomers that can be fermented

lactose → glucose and galactose → pyruvate

MR-VP broth

• two separate tests, but same exact medium (tested after incubation)

• MR: methyl red tests for mixed acid fermenters; 3-4 drops; detects low acid (< 4.4); positive stays red, negative turns yellow

• VP: vogues-proskauer; tests for 2,3-butanediol; barritt’s reagents (VP I and VP II) react w/ acetoin intermediate → acetoin; reacts w/ guanidine to produce red color

mixed acid v 2,3-butanediol

2,3-butanediol produces less acid and products are more easily converted to neutrality

(glucose → → acetoin → 2,3-butanediol)

baritt’s reagents

used in VP broth

VP I: alpha naphthol, intensifies red color

VP II: KOH, alkaline condition, favors acetoin oxidation → diacetal + O2, red color

tryptone broth

• detects tryptophanase: hydrolyze tryptophan → indole, pyruvate, + ammonia (NH3)

• 3-5 drops Kovac’s reagent (p-DMABA and HCl dissolved amyl or butyl alcohol); positive is pink top (cerise)

simmons citrate slant

• detects utilization of citrate as sole C source for growth, initially green

• indicator: bromothymol blue

• blue rxn almost always accompanies growth, but is not primary indicator, growth is

IMViC

set of four tests to differentiate E. coli from Enterobacter aerogenes

• indole (SIM or tryptone)

• methyl red

• voges proskauer

• citrate

dilution factor

A / (A+B)

A: volume transferred

B: volume being added to

plate dilution formulas

amount plated (mL) / 1 mL

dilutions for bacterial population counts

1. add 0.1 mL stock into 9.9 mL (1:100)

2. dilute 0.1 mL again into 9.9 mL (1:10,000)

3. do it once again (1:100,000)

4. 0.1 mL (1:10,000,000) and 1 mL (1:100,000) of final tube onto plates

5. 0.1 mL (1:100,000) and 1 mL (1:10,000) of 2nd tube onto plates

standard plate count

30 and 300 colonies in CFU/mL

which organism had negative results for phenol red sugar broth tests, which had all positives

negative: Pseudomonas aeruginosa

positive: E. coli, and gas production

which organism was able to degrade casein w/ proteolytic enzymes

Bacillus cereus, because there was a clearing

organism that caused a reversion in KIA slant

Enterobacter aerogenes, uses 2,3-butanediol fermentation