LABORATORY SUPPLY REQUEST
Microbiology 302
Winter, 2009
Note: Maintenance of positive and negative control organisms for staining reactions is the
responsibility of the students. Suitable media for transfer of stock cultures will be supplied upon
request.
Refer to the attached list of organisms for complete generic names.
DATE
1/6
(Chp 1,2)
MEDIA/SUPPLIES
PROVIDED/GROUP
Acid-fast stain reagents
Gram stain reagents
4 N.A. slants or plates
ORGANISMS
M. phlei or M. smegmatis
N. sicca or M. catarrhalis
S. aureus
B. subtilis
E. coli
Note: From this point on, for each group of organisms listed, each individual at your table
will be given one organism from the list as an unknown for inoculation onto appropriate
media. It is the responsibility of the entire table to develop flow charts to identify all of the
organisms listed and to turn in reports giving the identity of each of the unknowns at your
table. Included in the reports will be the flow charts that you developed. The reports will be
worth 10 points each, for a total of 80 points.
1/8
(Chp 4)
8 sets glucose Staph. O/F media
8 CBA
S. aureus
4 MSA (2 organisms/plate)
S. epidermidis
8 Rabbit plasma
M. luteus
4 DNAse plates (2 organisms/plate)
S. saprophyticus
4 Mueller-Hinton plates (100 mm; 2 organisms/plate)
Catalase
Sterile cotton swabs
Bacitracin (Taxo A) disks (.04 units)
Novobiocin disks, 5 mg
Mineral oil
37o C water bath or heating block
1/13
(Chp 4)
12 CBA plates (4 for CAMP – 2/plate)
8 Bile esculin slants
8 6.5% NaCl
S. pyogenes (Group A)
S. agalactiae (Group B)
E. faecalis (Group D)
S. bovis (Group D)
S. pneumoniae - smooth (rough demo)
(No Lancefield group)
PYR disks + reagents
Bacitracin (Taxo A) disks
Optochin (Taxo P) disks
1 N HCl in dropper bottle
S. aureus for CAMP test
Catalase reagent
10% Na desoxycholate (bile solubility test)
Sodium hippurate reagent (rapid test)
Candle jars
Staph/Micrococcus group unknowns due (10 points)
1/15
(Chp 5)
8 Choc.
8 CBA
4 T.M. (2 organisms/plate)
8 sets CarboFerm Neisseria Tests
N. meningitides
N. gonorrhoeae
N. lactamica
M. catarrhalis (B. catarrhalis)
Nitrate discs
B lactamase disks (cefinase)
Catalase reagent
Oxidase reagent
Candle jars
Strep. group unknowns due (10 points)
1/20
(Chp 7)
30 point quiz
8 Mac, 8 SS, 8 TSI, 8 CBA, 8 motility,
8 nitrate,16 MR-VP, 8 citrate, 8 PA,
8 Enteroscreen 4 or 8 urea + 8 LIA
E. coli
Shigella boydii (Group C)
K. pneumoniae
E. aerogenes
Oxidase reagent
Catalase reagent
Spot indole reagent
Nitrate disk reagents
Neisseria group unknowns due (10 points)
1/22
(Chp 7)
8 Mac, 8 HE, 8 CBA, 8 CIN, 8 TSI,
16 MR-VP, 8 PA, 8 motility, 8 citrate,
8 nitrate, 8 Enteroscreen 4 or 8 LIA +
8 urea
S. typhi
Salmonella sp. other than typhi
C.freundii
P. mirabilis
Y. enterocolita
Oxidase reagent
Nitrate reagents
Spot indole reagent
MR reagent
VP reagents
10% ferric chloride
Shigella polyvalent Group C typing serum
PYR disks + reagents (if Enteroscreen 4 is used)
Enterobacteriaceae I group unknowns due (10 points)
1/ 27
(Chp 7, 8)
8 Mac, 8 HE, 8 CBA, 8 CIN, 8 TSI,
16 MR-VP, 8 PA, 8 motility, 8 citrate,
6 ODC, 6 ADC, 6 nitrate, 6 urea, 6 LIA,
V. alginolyticus
A. hydrophila
P. multocida
Mineral oil
demo of C. jejuni
Oxidase reagent
(on choc + campy pak)
Nitrate reagents
Spot indole reagent
MR reagent
VP reagent
10% ferric chloride
PYR disks + reagents (if Enteroscreen 4 is used)
Salmonella polyvalent O and Salmonella Vi typing serum
Enterobacteriaceae II group unknowns due (10 points)
1/ 29
(Chp. 9)
30 point quiz
8 Mac, 8 CBA, 8 sets NF O/F Glucose media, A. lwoffi
8 motility, 8 TSI, 8 nitrate, 8 PA,
P. aeruginosa
8 rapid LDC, 8 rapid ODC
C. meningoseptica
S. maltophilia (X. maltophilia)
Mineral oil
Sxt. Disks
Oxidase reagent
Nitrate reagents
Spot indole reagent
MR reagent
VP reagents
10% Ferric chloride
Vibrio, et al. group unknowns due (10 points)
2/3
8 CBA (for streaking for isolation),
(Chp 6, 10) 4 CBA (2/plate for satellite testing),
8 Bile esculin, 8 BHI,
16 Motility (RT and 370 C)
8 Choc
X, V & XV strips
B lactamase (cefinase)
Bacitracin disks (10 units)
H. influenzae
H. parainfluenzae
L. monocytogenes
C. xerosis
G. vaginalis (demoon vaginalis agar)
S. aureus for satellite testing
10% Ferric chloride
Oxidase reagent
Nitrate reagents
Nonfermemter group unknowns due (10 points)
2/5
Midterm Exam (100 points)
Oxidase reagent
Catalase reagent
Haemophilus, et al. group unknowns due (10 points)
Fecal unknown 1 handed out; primary isolation media will be provided.
2/10
Work on fecal unknown; biochemical media will be provided upon justified request.
Blood unknown 2 handed out; primary isolation media will be provided.
2/12
Work on unknowns; biochemical media will be provided upon justified request
2/17
Work on unknowns; biochemical media will be provided upon justified request
Practice with micropipettors
Anaerobe educator:
Bacteriodes fragilis group
Bacteroides ureolyticus
Clostridium perfringens
Clostridium difficile
Fusobacterium nucleatum
Fusobacterium necrophorum
Peptostreptococcus anaerobius
Porphyromonas spp.
Prevotella intermedia
Propionibacterium acnes
2/19
Genus and species name for fecal unknown 1 due at beginning of class (30 points);
Work on unknown 2; biochemical media will be provided upon justified request
Anaerobe educator continued
Isolate DNA for molecular unknown; the instructor will determine the DNA
concentration. (Refer to DNA Isolation and DNA Concentration Determination
handouts)
Supplies required:
QIAamp DNA isolation kit
Micropipettors
Pipet tips
2 ml. microcentrifuge tubes
Microcentrifuge
370C, 560C, and 900C heating blocks or water baths
Lysis solution (20 mg/ml lysozyme, 20mM Tris-HCl pH 8.0, 2 mM EDTA,
1.2% Triton X-100)
Ethanol
Proteinase K (provided with kit)
Gloves
2/24
Genus and species name for blood unknown 2 due at beginning of class (30 points).
Anaerobe educator continued
Set up PCR for molecular unknown (refer to PCR handout)
Supplies required:
Unknown DNA (isolated by students during previous lab period)
Control DNA (previously isolated by the instructor)
Primers 1 (RW01) and 2 (dg74)
10X PCR buffer
dNTPs (200 uM)
Taq polymerase
Micropipettors
Pipet tips
PCR tubes
Ice in insulated containers
dH2O
Microcentrifuge
Thermocycler
Gloves
2/26
Agarose gel electrophoresis of PCR products (refer to Agarose Gel Electrophoresis
handout)
Cut DNA band from the gel and store in refrigerator until next lab period
Supplies required:
1% agarose gel (already prepared)
Power supply
0.5X TBE
100 basepair ladder
6X gel loading buffer
Micropipettors
Pipet tips
Razor blades
UV transilluminator
Film or versadoc
2 ml screw cap microcentrifuge tubes
Microcentrifuge
Gloves
3/3
Elute Molecular Unknown and Known DNAs from gel (refer to DNA Elution From
Agarose Gels handout)
Sequencing reaction (refer to Sequencing Reaction handout)
Supplies required:
Gel extraction kit (QIA quick)
500 C heating block
Isopropanol
Unknown and known DNAs (DNA you extracted from the gel, above)
Reaction premix
Sequencing buffer
Primer (RW01)
PCR tubes
Micropipettors
Pipet tips
Microcentrifuge
Thermocycler
Ice in insulated container
Gloves
3/5
Remove excess dyes in preparation of running sequencing gel (refer to DyeEx Spin Kit
handout)
Supplies required:
DyeEx spin kit
2 ml. microcentrifuge tubes
Microcentrifuge
Speed-vac dryer (lab to be determined)
Gloves
Set up PCR reaction to identify strains of E. coli carrying a shiga-like toxin (refer to PCR
handout)
Supplies required:
Unknown sample
Primers 1-4
10X PCR buffer
dNTPs (200 uM)
Taq polymerase
Micropipettors
Pipet tips
PCR tubes
Ice in insulated containers
dH2O
Microcentrifuge
Thermocycler
Gloves
3/10
Agarose gel of PCR products (refer to Agarose Gel Electrophoresis handout)
Supplies required:
1% agarose gel (already prepared)
Power supply
0.5X TBE
100 basepair ladder
6X gel loading buffer
Micropipettors
Pipet tips
Gloves
UV transilluminator
Film or versadoc
Analysis of agarose gel results to identify pathogenic E. coli strains
Analysis of sequencing results to identify Molecular Unknown (use BLAST databank)
3/12
FINAL EXAMINATION - 100 Points (given during lecture time)
Genus and species of molecular unknown due (50 points)