Partial Materials in the Final Lab
Exam (Nov. 28/29): Labs #9-23
(All labs after the first lab exam)
Please also read the review sheets I handed
out on Nov. 20 in lab. I will have office hour
M/Th: 9:15 a.m. - 10:15 a.m. at DH 553 or DH
543 (my lab)
LAB 9: Single Colony Isolation
Know how to obtain single colonies through the
“Streak for Isolation” on an agar plate technique.
Streak Plate technique
Lab #9
All in
NA
plates
Micrococcus luteus
Serratia marcescens
Staphylococcus aureus
Klebsiella rosea
Blood agar (BA) is a differential medium.
- Some bacteria produce an enzyme that is able to lyse RBCs –
this process is hemolysis.
- By growing bacteria on blood agar we can determine if the
bacteria produce hemolysin and thus lyse the RBCs.
- Blood agar is NA to which sheep RBCs have been added.
- If hemolysin is produced by the bacteria it will be secreted
into the medium and the RBCs will be lysed
(the medium will be clear rather than red).
- So presence of clearing around the bacterial growth indicates
hemolysis.
- Growth on BA differentiates between the hemolytic and
non-hemolytic bacteria.
Gamma hemolysis = No hemolysis
Alpha hemolysis = Partial
Beta hemolysis = Complete
Lab 10: Stock and Working Slants
• Why did we prepare a stock and a working stock slant
for the unknown?
• Why did we grow the unknown in different media and
under different conditions?
“Working” “Stock”
Lab 11: Simple Staining & Bacterial Smear
• Understand simple, negative, and positive staining.
• Know how to prepare a bacteria smear
Demos: simple stains of:
Neisseria (diplococci)
Pseudomonas (bacilli)
Lab 12: Differential Staining (Gram Stain)
• Know the entire Gram Staining
procedure and the function of each step.
• Know the Endospore procedure (in
Appendix, p. 121)
GRAM STAIN
E coli (Gm -)
Staph epidermidis (Gm+)
ENDOSPORE STAIN
See Appendix IV, p. 121
Outcome for endospore +
for Micr20
Bacillus megaterium
Bacillus anthracis
Clostridium tetani
Cell Arrangements:
Lab 13: Selective and Differential Media
• EMB: Eosin-Methylene Blue
a. Differential and selective properties.
b.Contains bile salts and the dyes eosin and methylene
blue; all inhibitory to Gram-positive bacteria (e.g.
Staphylococcus aureus).
c. Selects for Gram-negative bacteria (e.g. Escherichia.
coli).
d.Differentiates lactose fermenting (dark color with
metallic sheen) from non-lactose-fermenting
(colorless) bacteria.
Salmonella pullorum
Staph. epidermidis
E. coli
Staph. aureus
Lab 13: Selective and Differential Media
TGA: Tellurite Glycine Agar
a.Selects for coagulase-positive staphylococci.
b.Differential: coagulase-positive cocci form black
colonies.
c.Coagulase-negative cocci are generally inhibited.
The ones that grow are gray.
d.Most Bacilli and Pseudomonas (Gm+) are inhibited.
e.Proteus sp rarely grows and form brown colonies.
Staph. aureus
Staph. epidermidis
E. coli
Salmonella pullorum
Know all the media we covered in Micro20 since lab #9:
The purpose of the medium, how to read a positive and
a negative result, what those results mean, and the
MAJOR components of the medium.
Lab 14: Antibiotic Sensitivity
• Antibiotics are chemicals that are produced by other bacteria/fungi
that have the ability to prevent other organisms (bacteria) from
growing or killing them.
• Sensitivity X Resistance to antibiotics.
• Bacteriostasis (stopping bacterial growth) X bacteriocide (killing
of bacteria).
• Broad spectrum antibiotics- effective against a wide range of
bacteria (G+ and G-).
• Narrow spectrum antibiotics - effective against a small specific
group of bacteria (either G+ or G-).
Lab 14: Antibiotic Sensitivity; Disc Diffusion Method
LAB 15: Catalase, Amylase, Gelatinase
(Proteinase), MRVP
• MRVP, see Appendix IV, p.118-119
CATALASE
Negative
Enterococcus faecalis
H2O2
Positive
Streptococcus aureus
Amylase: Starch Hydrolysis
BEFORE
E.coli
AFTER
Flood with
Iodine solution
Bacillus subtilis
E coli - (neg.)
Bacillus subtilis + (pos.)
Gelatinase test: Plate was flooded with Frazier’s Developer
Gelatinase +
Gelatinase
negative
LAB 16: Urease, SIM agar, Citrate
UREASE
• UREASE TEST: Urease is an enzyme that
breaks the carbon-nitrogen bond of amides
(e.g. urea) to form carbon dioxide,
ammonia, and water. Members of genus
Proteus are known to produce urease.
When urea is broken down, ammonia is
released and the pH of the medium
increases (becomes more basic). This pH
change is detected by a pH indicator that
turns pink in a basic environment. A pink
medium indicates a positive test for urease.
SIM agar
• SIM = Sulfide, Indole, Motility.
• INDOLE TEST: Indole is a component of the
amino acid tryptophan. Some bacteria have
the ability to break down tryptophan for
nutritional needs using the enzyme
tryptophanase. When tryptophan is broken
down, the presence of indole can be detected
through the use of Kovacs' reagent. Kovac's
reagent, which is yellow, reacts with indole and
produces a red color on the surface of the test
tube.
SIM agar
MOTILITY
Motile bacterium
Non-motile bacterium
(e.g. Pseudomonas aeruginosa)
(e.g. Staph aureus)
Citrate: The Simmon’s Citrate medium tests the ability of the
bacteria culture to be able to use citrate as the sole C source.
Bacteria that are able to produce the enzyme citrase are able to
transport the citrate into the cell and use it as a source of C.
Since the medium does not contain any other source for C, only
those bacteria that can produce citrase are able to grow in this
medium.
When cultures are able to use the citrate they break it down,
producing sodium bicarbonate, which changes the pH of the
medium to alkaline.
The pH indicator in the medium (bromothymol blue) changes to a
blue color from its original green color.
+
LAB 17: Carbohydrate Utilization
Lab 17: KIA medium
• C = Uninoculated
• 1 = Non-fermenter
• 2 = Glucose fermenter
3 = Glucose fermenter + H2S producer
4 = Glucose+Lactose fermenter, gas
5 = Gluc + Lact ferm + H2S producer
Lab 17: Fermentation of Carbohydrates
F- tubes
SUGAR Fermentation
Detection is based on acid production due to sugar fermentation. The pH indicator is
PHENOL RED. Phenol red turns yellow under acidic conditions. Hence, yellow
means a positive result.
The ability to ferment specific sugars is dependant on the ability of the
bacterium to produce the specific enzymes required for the
transport and metabolism of that particular sugar.
Thus fermentation of various sugars can be used to characterize bacteria.
The F-tubes use phenol red in the medium as pH indicator and the use
of inverted tubes to detect production of gases.
Results are recorded as Negative (no metabolism); Acid (+ reaction);
Acid + Gas (+ with gas production).
LAB 18: Unknown & Single Colony Isolation
Know how to use the Dichotomous Key to identify a
bacterium based on morphology, Gram staining,
endospore production, and various metabolic
reactions. See p.58-61of lab manual.
LAB 19: Pour Plate
•Pour Plate Technique
•Serial Dilution
•Colony Forming Unit (CFU)
•Quantification of Bacteria in Cell/ml
Lab 19: Pour Plate
• Bacteria Enumeration
1
2
3
4
5
Dilution Series:
1ml
100
(1:1
1ml
1ml
1ml
*Best 30-300 CFU
•CFU = 100
• Dilution = 1000
10-1 10-2 10-3 10-4 (Dilution)
1:10 1:100 1:1000 1:10,000)
•Hence 100 X 1,000 =
100,000 = 1x105
TMTC 1000 400
100*
20 (CFU)
Bacteria Enumeration
1x10-5
1x10-6
Cell /ml= (CFU X dilution factor) / volume
LAB 20: Most Probable Number
• MPN method, MPN table
• Durham tubes
• Presumptive, Confirmed, and Completed tests
Lab 20: Most Probable Number (MPN)
MPN method:
1st- Presumptive test:
growth on lauryl tryptose
broth
2nd - Confirmed test: on
Eosine-Methylene Blue Agar
(EMB)
3rd - Completed tests
Bacteria Enumeration (Presumptive)
LAB 21: Phage Characterization and
Quantification
• Plaque, Plaque Forming Unit (PFU)
• Serial Dilution, Phage quantification
• T1phage
BACTERIOPHAGE
1x10-4 Dilution
Plaque
(clear zone)
1x10-6 Dilution
LAB 22: Bacterial Aggutination &
Immunoprecipitation
•
Immunoprecipitation
- is the reaction between a soluble antigen and its specific antibodies
-soluble antigens are smaller and in solution; complexing with antibodies make th
bigger and they fall out of solution as a precipitate –visible to the eye.
Antibody specificity known (toxin, protein, etc.)
Antigen presence or identity not known (?)
Precipitationreaction between antibody and soluble antigen
Immunoprecipitation
Antibody specificity known (toxin, protein, etc.)
Antigen presence or identity not known (?)
Precipitationreaction between antibody and soluble antigen
Immunoprecipitation
Immunoprecipitation
Reaction
of
identity
Reaction of nonidentity
LAB 23:Staphyloslide Latex Test Kit
• Example of Agglutination
Bacterial agglutination
agglutination
no agglutination
S. aureus
S. epidermidis
?
Bacterial agglutination (new procedure).
#1 Mark your bacterial agglutination cards
S. aureus
S. epidermidis
Bacterial agglutination
#2 Mix the latex agglutination reagent dropper bottle
and dispense one drop onto each circle
S. aureus
S. epidermidis
Bacterial agglutination
#3 Using a sterile toothpick , pick up and smear 1 suspect
colony from your negative control in the proper ring.
#4 Using a NEW sterile toothpick , pick up and smear 1 suspect
colony from your positive control in the proper ring.
#5 Using a NEW sterile toothpick , pick up and smear 1
suspect colony from your unknown in the proper ring.
S. aureus
S. epidermidis
Bacterial agglutination (Part B).
#6 Pick up and gently rock the card for 20 seconds and
observe for agglutination under normal lighting
conditions
S. aureus
S. epidermidis
?
Bacterial antigen:
S. aureus
S. epidermidis
unknown
Agglutination with anti-S. aureus :