JAMES LUNSFORD.COM

STUDY GUIDE FOR MICROBIOLOGY FINAL

202.03 SPRING 2005

USE THIS DATA TO REVIEW THE FOLLOWING.

EXERCISE 30 ULTRAVIOLET LIGHT: LETHAL EFFECTS

EXERCISE 34 EVALUATION OF ANTISEPTICS AND DISINFECTANTS

EXERCISE 32 EVALUATION OF ALCOHOL

EXERCISE 33 ANTIMICROBIC SENSITIVITY TESTING

MINIMAL INHIBITORY CONCENTRATION

REVIEW HANDOUT

Antimicrobial sensitivity testing

Kirby Bauer method using a Mueller-Hinton II agar plate
Procedure: Measure zones of inhibition → determine which antibiotics your organism is sensitive (S), resistant (R) or intermediate (I).

Minimal Inhibitory Concentration

MIC is the lowest concentration of an antibiotic that inhibits the growth of a particular microorg

RADIAL IMMUNODIFFUSION (READ HANDOUT)

Can quantitatively determine the concentration of an antigen used clinically to detect patients levels of blood proteins
Procedure: antibody + agar → wells cut out of agar → known concentrations of antigen put in wells → measure antigen-antibody precipitate → graph

***refer to James’ notes***

EXERCISE 59 BLOOD GROUPING (READ HANDOUT, GENECTICS OF BLOOD

ELISA REACTIONS REVIEW HANDOUTS

Step one: antigen added to wells
Step two: solution that may/may not contain primary antibody is added (if present, primary antibody will bind to antigen and remain after washing)
Step three: solution with secondary antibody added (if primary antibody remained in well, secondary antibody will bind to it and remain after washing) secondary antibodies are purified and covalently cross linked to horseradish peroxidase, wash away unbound secondary antibody
Step four: Substrates 1 added to wells 1 and 2, substrate 2 added to wells 3 and 4 (contains hydrogen peroxide)

***refer to James’ notes***

TO VIEW A CHART OF UNKNOWN #1

Examination of unknown colonies:

STAPH usually larger than STREP color usually opaque (white or yellow)

STREP usually very small color is pale/translucent

**EXCEPTION is S. Pneumoniae à colonies tend to be mucoid and have a pale, fried egg appearance

MICROCO usually smaller than STAPH and yellow color

STAPH vs STREP

Unknown #1

Bacteria	*Shape*	*Gram*	*Arrangement*
Staphylococcus aureus	cocci	+
Staphylococcus epidermidus	cocci	+
Micrococcus luteus	cocci	+
Streptococcus pneumonia	cocci	+
Streptococcus pyogenes	cocci	+
Enterococcus faecalis	cocci	+

Test Performed on Cocci, gram +, bacteria AND PICS

by AUDREY ESTRADA (GREAT JOB!!!)

Name of test	Description and Procedures	results
Catalase	· Procedure: glass slide → loop of org → 1 drop H2O2 → results immediate (bubbles/no bubbles) · Catalase = inactivation of H2O2	· Bubbles: staph, bacillus or micrococci No bubbles: entercocci or strept
MSA	Procedure: inoculate MSA plate ONLY STAPH WILL GROW Strept and micrococci will not grow Mannitol fermentation → yellow zone → acid (pH lowers)	Growth: +/- Acid (yellow): +/-
Coagulase	Procedure: inoculate tube with rabbit citrate plasma (citrate prevents coagulation) TARGETS S. AUREUS ONLY	+ if solidification (clotting) of rabbit citrate plasma occurs (only for S. aureus)
ά hemo test: optochin	Procedures: swab ½ blood agar plate → place OP ID → put in candle jar	+ (susceptible): zone of inhibition - : no change
ά hemo test: Salt tolerance	Procedures: inoculate 6.5% NaCl tube → incubate	Growth +/-
ά hemo test: Bile esculin	Procedures: stab broth to bottom → zigzag on surface → incubate FOR ENTERCOCCI Inhibits many gram + Cleavage of eskulin to eskuletin in presence of ferric citrate= black	+: black
ά hemo test: Bile solubility	if an alpha hemo strept organism is soluble in bile and + on optochin test → S. pneumoniae
β hemo test: Bacitracin susceptible	Procedures: streak unknown heavily over 40% of plate → draw line downward → inoculate a line 1 cm away from unknown of S. aureus → place disk in 40% Test for S. pyogenes	+: zone of inhibition
β hemo test: SXT	Procedures: place disk alongside bacitracin disk Identify beta hemo strept	R: resistant S: susceptible (zone of inhibition)

Catalase Optochin

MSA Bile esculin

Blood Agar plates

Hemolysis

Alpha hemolysis: this is the partial destruction of red blood cells, and often has a greenish hue to it rather than an actual clearing around the colonies,
Beta hemolysis: which is the complete destruction of red blood cells, usually, if you can see a finger or some other object through the clearing on the plate
Gamma hemolysis: no hemolysis

Professor Ammons http://jameslunsford.com/MICRO202_EX8_10_11.html

UNKNOWN # 2 - 8 Possibilities as follows

Page 335 Exercise 54 says: SALMONELLA & SHIGELLA are enteric pathogens of prime medical concern. They cause enteric fevers, food poisoning and bacillary dysentery. SALMONELLA TYPHI causes typhoid fever and is by far the most significant pathogen in the SALMONELLA group.

SHIGELLA, which is the prime cause of human dysentery, comprises of four species and many serotypes.

This experiment is attempting to demonstrate how to isolate your normal micro biota intestinal flora from invaders like SALMONELLA & SHIGELLA. If you have a POSITIVE LACTOSE TEST RESULT there could be CITROBACTER, ENTEROBACTER, ESCHERICHIA, KLEBSIELLA. Conversely, if you get a NEGATIVE LACTOSE test result there could be PROTEUS, PSEUDOMONAS, SALMONELLA, and SHIGELLA. There are obviously more tests that indicate specificity of your unknown and you may examine your results to isolate your organism.

By looking at the organizational chart it says that if your unknown LACTOSE NEGATIVE, GLUCOSE POSITIVE, NONMOTILE AND H2S- (NEGATIVE)…. YOU GOT THE FUNK (SHIGELLA). If the LACTOSE NEGATIVE, GLUCOSE POSITIVE, MOTILE AND H2S- NEGATIVE, and UREA NEGATIVE…YOU STILL GOT THE FUNK (SALMONELLA).

IF ORGANISM IS LACTOSE NEGATIVE, GLUCOSE POSITVE, NONNOTILE AND HYDROGENSULFIDE POSITIVE (BLACK)-THINK SHIGELLA! (Which is the prime cause of human dysentery, comprises of four species and many serotypes).

IF ORGANISM IS LACTOSE NEGATIVE, GLUCOSE NEGATIVE, THINK PSUEDOMONAS.

IF ORGANISM IS LACTOSE NEGATIVE, GLUCOSE POSITVE, MOTILE…PERFORM UREA TEST AND IF IT IS UREA POSITVE ORGANISM IS PROTEUS ON THE CONTRARY IF THERE IS A NEGATIVE UREA TEST RESULT…SALMONELLA! (TYPHI causes typhoid fever and is by far the most significant pathogen in the SALMONELLA group.

By ANGELICA

SALMONELLA & SHIGELLA

- cause enteric fevers, food poisoning, & bacillary dysentery

- lactose fermentation separates salmonella & shigella from most of the

other Enterobacteriaceae (they are both non-lactose fermenting)

- most salmonella grow unrestricted on selenite F & gram negative

broths while shigella are inhibitedt o some extent in selenite F broth

- salmonella is motile & shigella is non-motile

- On MacConkey agar Salmonella, Shigella, & other non-lactose

fermenting species produce smooth, colorless colonies.

- On Hektoon Enteric (HE) agar Salmonella & Shigella colonies are

greenish-blue. Some species of Salmonella will have greenish-blue

colonies w/ black centers due to H₂S production.

- On Xylose Lysine Desoxycholate (XLD) agar, although most Salmonella

produce red colonies w/ black centers, a few may produce red colonies

that lack black centers. Shigella colonies are red.

figure 54.1 in your lab manual, above, will not appear until I have converted to html and uploaded org chart.

Bacteria	Shape	Gram	Arrangement
CITROBACTER	rod	-
ENTEROBACTER	rod	-
ESCHERICHIA	rod	-
KLEBSIELLA	rod	-
PROTEUS	rod	-
PSEUDOMONAS	rod	-
SALMONELLA	rod	-
SHIGELLA	rod	-

Name of test	Description and Procedures	Results
EMB (eosin methylene blue)	Selective: inhibits gram+ Differential: fermenter/non fermenter	Lactose +: purple +/- green sheen Lactose -: pale
SS	Selective: inhibits gram+ Differential: fermenter/ non fermenter	Lactose +: bright pink Lactose -: pale H2S: black
Oxidase	Procedures: filter disk on slide → soak paper with oxidase → streak loopful of bacteria on disk Tests for the presence of cytochrome oxidase	+: blue/purple: pseudomonas -: no color: enterbacter
Motility	Procedure: stab semisolid agar with tetrasodium salt	+: stab spreads -: no spreading of bacteria
Citrate	Procedures: zig zag on surface of slant Determine whether org uses citrate as a sole source of carbon	+: turns blue -: remains green
Indole	Procedures: inoculate tube → incubate 24 hrs → 3 drops KOVACS reagent Determine if org can split the amino acid tryptophan into indole and pyruvic acid	+: red layer at the top of broth
Urea	Procedures: inoculate tube and incubate This test is used to differentiate organisms based on their ability to hydrozye urea with the enzyme urease. Useful in identifying genera PROTEUS	+: turns pink
Methyl Red	Procedures: inoculate tube of MR-VP broth and incubate Used to identify bacteria that produce stable acid end products by means of mixed acid fermentation of glucose.	+: red
VP: Voges-Proskauer	Procedures: same as Methyl red This test is used to identify organisms able to produce acetoin from the degradation of glucose during a 2,3-butanediol fermentation.	+: red
TSI (triple sugar iron)	Procedures: stab and streak TSI slant Refer to media used for more info	Sugars fermented → acid → yellow Peptones used → pH rise → pink H2S+: black ppt H2S-: no ppt

Oxidase

Motility

Citrate

Indole

Urea

Methyl red

TSI

Coliforms

Definition: gram –, facultative anaerobic, non-endospore forming rods that ferment lactose to produce acid and gas
Lactose fermentation with the formation of acid and gas provides the basis for determining the total coliform count of water samples
E.coli (coliform) is a good indicator of fecal contamination because

Found in human intestine and some warm-blooded animals but not in soil or water
Easily identified in microbio tests
Not picky as the intestinal pathogens so it survives a little longer

Other indicators: E.coli and Enterobacter aerogenes (coliforms) and Streptococcus faecalis (gram+ enterococcus)

Media and Reagents Used

Mueller-Hinton II agar: general (?)

used in antimicrobial sensitivity testing (Kirby Bauer test)

Rabbit citrate plasma: differential (?)

coagulase test for S. aureus in unkwn #1

TSI (triple sugar iron) slant: differential (?)

Contains 1% lactose 1% sucrose .1% glucose
Also contains peptone, phenol red (pH indicator: yelow at pH less than 6.8 and red above 6.8)
thiosulfate (sulfur source), ferric ammonium/citrate
if sugars are fermented → acid → yellow
if peptones used → amine → pH rise → pink

EMB (eosin methylene blue): selective and differential

selective: bile salts, eosin, methylene blue → inhibits growth of gram +
differential: lacoste → fermenter/nonfermenter

SS: selective and differential

1. selective: bile salts, sodium citrate, brilliant green → inhibits gram +

2. differential: same as EMB

MR-VP broth: differential (?)

1. includes peptone, glucose, and a phosphate buffer

Barritt's Reagent A (alpha-napthol) and Barritt's Reagent B

1. VP test reagents

Kovac’s reagent

used for tryptone (indole) test

EXERCISE 47 BACTERIOLOGICAL EXAMINATION OF WATER:

QUALITATIVE TESTS

Three tests done: presumptive (look for gas), confirmed

(streak EMB → gram stain → look for gram-), and completed

DO NOT RELY ON THIS FOR YOUR LAB FINAL!!!!