LABORATORY SUPPLY REQUEST
Microbiology 302
Winter, 2012
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
MEDIA/SUPPLIES
PROVIDED/GROUP
1/10
Acid-fast stain reagents
(Chp 1,2,12) 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
(up to six students) 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.
1/12
(Chp 4)
6 sets glucose Staph. O/F media
S. aureus
6 CBA
S. saprophyticus
3 MSA (2 organisms/plate)
S. epidermidis
6 Rabbit plasma
M. luteus
3 DNase plates (2 organisms/plate)
3 Mueller-Hinton plates (100 mm; 2 organisms/plate)
Catalase
Sterile cotton swabs
Bacitracin Taxo A disks (0.04 units)
Novobiocin disks, 5 ug
Mineral oil
37o C water bath or heating block
1/17
(Chp 4)
8 CBA plates (2 for CAMP – 3/plate)
6 Bile esculin slants
6 - 6.5% NaCl
PYR disks + reagents
Bacitracin Taxo A disks
Optochin Taxo P disks
1 N HCl in dropper bottle
Catalase reagent
10% Na desoxycholate (bile solubility test)
Sodium hippurate reagent (rapid test)
Candle jars
S. pyogenes (Gp. A)
S. agalactiae (Gp. B)
S. pneumoniae ( - )
E. faecalis (Gp. D)
S. bovis (Gp. D)
S. aureus for CAMP test
1/19
(Chp 5)
Staph/Micrococcus Group Unknown Report due (10 points)
6 Choc.
N. meningitidis
6 CBA
N. gonorrhoeae
3 T.M. (2 organisms/plate)
N. lactamica
6 sets chromogenic enzyme reactions
M. catarrhalis
(CarboFermTMNeisseria Kit)
Nitrate discs
B lactamase disks (cefinase)
Catalase reagent
Oxidase reagent
Candle jars
1/24
(Chp 7)
Strep Group Unknown Report due (10 points)
6 Mac, 6 SS, 6 TSI, 6 CBA, 6 motility,
6 nitrate,12 MR-VP, 6 citrate, 6 LIA,
6 urea, 6 PA
E. coli
Shigella sonnei
E. aerogenes
K. pneumoniae
Mineral oil
Oxidase reagent
Catalase reagent
Spot indole reagent
Nitrate disk reagents
1/26
(Chp 7)
Neisseria Group Unknowns Report due (10 points)
20 point quiz
6 Mac, 6 HE, 6 CBA, 6 TSI, 12 MR-VP,
S. typhi
6 PA, 6 motility, 6 citrate, 6 LIA, 6 urea,
P. mirabilis
6 nitrate, 6 CIN
C. freundii
Y. enterocolitica
Mineral oil
Oxidase reagent
Nitrate reagents
Spot indole reagent
MR reagent
VP reagents
10% ferric chloride
Shigella typing serum
1/ 31
(Chp 7, 8)
Enterobacteriaceae I Group Unknown Report due (10 points)
6 Mac, 6 HE, 6 CBA, 6 CIN, 6 TSI,
V. alginolyticus
12 MR-VP, 6 PA, 6 motility, 6 citrate,
A. hydrophila
6 LIA, 6 ODC, 6 ADC, 6 urea, 6 nitrate,
P. multocida
Mineral oil
Oxidase reagent
Nitrate reagents
Spot indole reagent
MR reagent
VP reagent
10% ferric chloride
Salmonella typing serum
Demo of C. jejuni
(on choc + campy pak)
2/2
(Chp. 9)
Enterobacteriaceae II Group Unknown Report due (10 points)
6 Mac, 6 CBA, 6 sets NF O/F Glucose media,
A. lwoffi
6 motility, 6 TSI, 6 nitrate, 6 PA,
P. aeruginosa
6 rapid LDC, 6 rapid ODC
S. maltophilia
C. meningoseptica
Mineral oil
Sxt. Disks
Oxidase reagent
Nitrate reagents
Spot indole reagent
MR reagent
VP reagents
10% Ferric chloride
2/7
(Chp 6, 10)
Vibrio et al. Group Unknown Report due (10 points)
6 CBA (for streaking for isolation),
H. influenzae
3 CBA (2/plate for satellite testing),
H. parainfluenzae
6 Bile esculin, 4 BHI,
L. monocytogenes
0
12 Motility (RT and 37 C)
C. xerosis
6 Choc
S. aureus for satellite testing
X, V & XV strips
B lactamase (cefinase)
G. vaginalis (demoBacitracin disks (10 units)
on vaginalis agar)
10% Ferric chloride
Oxidase reagent
Nitrate reagents
2/9
2/14
Midterm Exam (100 points)
Nonfermenter Group Unknown Report due (10 points)
Oxidase reagent
Catalase reagent
Fecal Unknown handed out; primary isolation media will be provided.
Haemophilus et al. Group Unknown Report due (10 points)
Work on Fecal Unknown; biochemical media provided upon justified request.
Blood Unknown handed out; primary isolation media will be provided.
2/16
Work on Unknowns; biochemical media provided upon justified request.
2/21
Continue work on Unknowns
Practice with micropipettors
Anaerobe educator: Gram stain reaction & morphology, size, clinical significance
Bacteriodes fragilis group
Bacteroides ureolyticus
Clostridium perfringens – continued on next page
Clostridium difficile
Fusobacterium nucleatum
Fusobacterium necrophorum
Peptostreptococcus anaerobius
Porphyromonas spp.
Prevotella intermedia
Propionibacterium acnes
2/23
Genus-species for Fecal Unknown Report due at beginning of class (20 points)
Anaerobe educator continued
Isolate DNA from 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
2/28
Genus-species for Blood Unknown Report due at beginning of class (20 points)
Anaerobe educator continued
Set up PCR for Molecular Unknown DNA (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
3/1
Agarose gel electrophoresis of Molecular Unknown PCR product (refer to Agarose
Gel Electrophoresis handout)
Cut Molecular Unknown 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 DNA ladder
5X gel loading buffer
Micropipettors
Pipet tips
Razor blades
UV transilluminator
Film or versadoc
2 ml screw cap microcentrifuge tubes
Microcentrifuge
Gloves
3/6
Elute Molecular Unknown DNA from gel (refer to DNA Elution from Agarose Gel
Handout)
Sequencing reaction (refer to Sequencing Reaction handout)
Supplies required:
Gel extraction kit
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
3/8
Remove excess dyes in preparation of running sequencing gel for Molecular Unknown
DNA (refer to DyeEX Spin Kit handout)
Supplies required:
DyeEx Spin Kit
2 ml. microcentrifuge tubes
Microcentrifuge
Speed-vac dryer (McQueen lab)
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
3/13
Agarose gel of E. coli PCR products (refer to Agarose Gel Electrophoresis handout)
Supplies required:
1% agarose gel (already prepared)
Power supply
0.5X TBE
100 basepair DNA ladder
5X gel loading buffer
Micropipettors
Pipet tips
Gloves
UV transilluminator
Versadoc to record gel DNA bands
Analysis of agarose gel results to identify pathogenic E. coli strains
Analysis of sequencing results to identify Molecular Unknown (use BLAST databank)
3/15
LABORATORY FINAL EXAM - 100 Points
Genus-species of Molecular Unknown Report due (40 points)