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)

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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 915 a.m. - 1015 a.m. at DH 553 or DH
543 (my lab)
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LAB 9 Single Colony Isolation
Know how to obtain single colonies through the
Streak for Isolation on an agar plate technique.
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Streak Plate technique
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Lab 9
Micrococcus luteus
Staphylococcus aureus
All in NA plates
Klebsiella rosea
Serratia marcescens
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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.
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Gamma hemolysis No hemolysis Alpha hemolysis
Partial Beta hemolysis Complete
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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?

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Working Stock
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Lab 11 Simple Staining Bacterial Smear

• Understand simple, negative, and positive
  staining.
• Know how to prepare a bacteria smear

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Demos simple stains of Neisseria (diplococci)
Pseudomonas (bacilli)
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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)

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GRAM STAIN
E coli (Gm -)
Staph epidermidis (Gm)
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ENDOSPORE STAIN
Bacillus megaterium
See Appendix IV, p. 121
Outcome for endospore for Micr20
Bacillus anthracis
Clostridium tetani
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Cell Arrangements
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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.

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Salmonella pullorum
E. coli
Staph. epidermidis
Staph. aureus
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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.

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E. coli
Staph. aureus
Staph. epidermidis
Salmonella pullorum
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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.
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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-).

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Lab 14 Antibiotic Sensitivity Disc Diffusion
Method
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LAB 15 Catalase, Amylase, Gelatinase
(Proteinase), MRVP

• MRVP, see Appendix IV, p.118-119

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CATALASE
H2O2
Negative
Positive
Enterococcus faecalis
Streptococcus aureus
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Amylase Starch Hydrolysis
BEFORE
AFTER
Flood with Iodine solution
E.coli
Bacillus subtilis
E coli - (neg.) Bacillus subtilis (pos.)
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Gelatinase test Plate was flooded with Fraziers
Developer
Gelatinase
Gelatinase negative
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LAB 16 Urease, SIM agar, Citrate
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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.

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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.

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SIM agar
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MOTILITY
Motile bacterium
Non-motile bacterium
(e.g. Staph aureus)
(e.g. Pseudomonas aeruginosa)
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Citrate The Simmons 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.

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LAB 17 Carbohydrate Utilization
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Lab 17 KIA medium

• C Uninoculated 3 Glucose fermenter H2S
  producer
• 1 Non-fermenter 4 GlucoseLactose fermenter,
  gas
• 2 Glucose fermenter 5 Gluc Lact ferm H2S
  producer

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Lab 17 Fermentation of Carbohydrates F-
tubes
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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.
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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).
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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.
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LAB 19 Pour Plate

• Pour Plate Technique
• Serial Dilution
• Colony Forming Unit (CFU)
• Quantification of Bacteria in Cell/ml

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Lab 19 Pour Plate

• Bacteria Enumeration

• Dilution Series
• 100 10-1 10-2 10-3 10-4 (Dilution)

1ml
1ml
1ml
1ml
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Bacteria Enumeration
1x10-5
1x10-6
Cell /ml (CFU X dilution factor) / volume
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LAB 20 Most Probable Number

• MPN method, MPN table
• Durham tubes
• Presumptive, Confirmed, and Completed tests

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Lab 20 Most Probable Number (MPN)
Bacteria Enumeration (Presumptive)
MPN method
1st- Presumptive test growth on lauryl tryptose
broth
2nd - Confirmed test on Eosine-Methylene Blue
Agar (EMB)
3rd - Completed tests
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LAB 21 Phage Characterization and Quantification

• Plaque, Plaque Forming Unit (PFU)
• Serial Dilution, Phage quantification
• T1phage

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BACTERIOPHAGE
1x10-4 Dilution
Plaque (clear zone)
1x10-6 Dilution
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LAB 22 Bacterial Aggutination
Immunoprecipitation

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• Immunoprecipitation
• - is the reaction between a soluble antigen and
  its specific antibodies
• soluble antigens are smaller and in solution
  complexing with antibodies make these
• 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
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Immunoprecipitation
Antibody specificity known (toxin, protein,
etc.) Antigen presence or identity not known
(?) Precipitation?reaction between antibody and
soluble antigen
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Immunoprecipitation
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Immunoprecipitation
Reaction of identity
Reaction of nonidentity
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LAB 23Staphyloslide Latex Test Kit

• Example of Agglutination

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Bacterial agglutination
agglutination
no agglutination
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Bacterial agglutination (new procedure).
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Mark your bacterial agglutination cards
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Bacterial agglutination
2
Mix the latex agglutination reagent dropper
bottle and dispense one drop onto each circle
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Bacterial agglutination
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Using a sterile toothpick , pick up and smear 1
suspect colony from your negative control in the
proper ring.
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Using a NEW sterile toothpick , pick up and smear
1 suspect colony from your positive control in
the proper ring.
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Using a NEW sterile toothpick , pick up and smear
1 suspect colony from your unknown in the proper
ring.
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Bacterial agglutination (Part B).
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Pick up and gently rock the card for 20 seconds
and observe for agglutination under normal
lighting conditions

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