Microbiology 2420 Practical 2 (collin college dr. cain's class)

1. Microbiology 2420
Practical 2
Ilana Kovach

2. Identification of
Bacillus Species

3. Starch media
Substrate:
Polysaccharide Starch
Enzyme:
Amylase & Maltase
starch
+
+
-
-Maltose
(disaccharide)
Glucose
(monosaccharide)
Amylase Maltase

4. Lipid media
Substrate:
Tributyrin
Enzyme:
Lipase
Triglycerides
+
+ (Slightly)
+
-
Glycerol
Fatty acids

5. Caesin Media
Substrate:
Caesin
Enzyme:
Protease & Casease
Casein
+
+
+ -
Peptones Polypeptides dipeptides
Amino
acids

6. +
+
+ - -
-

7. Gelatin media
Substrate:
Gelatin (Protein produced by hydrolysis of Collagen)
Enzyme:
Gelatinase
Gelatin
+ -
Amino acid components
“cause gelatin to liquefy”
Gelatinase

8. Mannitol Salt agarSelective (7.5% NaCl)
halotolerant
Differential (Mannitol)
pH indicator:
phenol Red
Red  Yellow

9. Nitrate ReductionDifferential Only (Beef extract, Peptones & Nitrate)
Enzyme:
Nitrate Reductase
Anaerobically reduce nitrate  nitrite  Ammonia or completely to Nitrogen
(5 drops of Each)
Nitrate A = Sulfanilic Acid
Nitrate B = Alpha-naphythylamine
Yellow Positive
(nitrates reduced beyond
nitrites to ammonia or
molecular nitrogen)
RED Positive
reduced Nitrate  Nitrite
Yellow Add Zinc
RED Negative
(The zinc reduced
the nitrate NOT the
bacteria)

10. Citrate UtilizationSelective Only (Sodium Citrate, Sodium & Water)
Enzyme:
Citrase
pH indicator:
Bromothymol Blue
CO2
Sodium
citrate
Oxaloactetic
acid
Acetic
acid
pyruvate
Alkaline
Sodium
bicarbonate
pH
Indicator
Reacts
with
Na+ & H2O
Reacts
with

11. Bile Esculin (BEA)
Selective (Bile Salts)… Gram Negative MacConkey
Differential (Esculin & Ferric ammonia Citrate)
Hydrolyze the carbohydrate Esculin
Esculin
EsculetinGlucose
Ferric Ammonia Citrate
“form black precipitate”
Reacts
with

12. Transformation

13. Escherichia coli + pGreen Plasmid  Fluorescent Escherichia Coli
*CaCl (increase permeability)
Competent Cells
Artificially competent
Vector
Plasmid vectors engineered to contain
1) Selectable marker
2) Cloning site
Transformation Efficacy
# 𝒐𝒇 𝒕𝒓𝒂𝒏𝒔𝒇𝒐𝒓𝒎𝒂𝒕𝒂𝒏𝒕𝒔
𝒂𝒎𝒐𝒖𝒏𝒕 𝒐𝒇 𝑫𝑵𝑨 (𝒎𝒊𝒄𝒓𝒐𝒈𝒓𝒂𝒎𝒔)
Transformation
LB Before: Growth
LB after: Growth
LBamp Before: No growth
“NOT resistant to ampicillin”
LBamp After: Growth
“Resistant to ampicillin” &
Glows

15. identification of
Staphylococcus
Species

16. Catalase Test
H2O2 (Hydrogen Peroxide) 2H20 (Water) + O2 (Oxygen)
Staphylococcus +
Streptococcus/
Enterococcus -

17. Mannitol Salt Agar
Selective (7.5% NaCl) halotolerant
Differential (Mannitol)
pH indicator:
phenol Red
Red  Yellow
+
+
weak
+
-

18. DNase agar
Differential: Methyl green dye &
polymerized DNA form complex
which give the agar a Blue-Green
Color at pH 7.5
DNA
smaller
fragments
unbound to
methyl green
resulting in a zone of clearing
+ +
- -

19. Mueller Hinton AgarTest Susceptibility of Novobiocin
>18mm
Nutrient agar (Neither
selective or differential)
Controlled: Levels of thymine, Thymidine, Ca+ ions & Mg
+
+
-
-

20. Rapid Staph Test
Latex particles
coated with human fibrinogen & IgG
Staphylococcus aureus
Particles will bind to any staph bacteria that
Contain coagulase/Protein A
“Protein A bind to Fc region of IgG antibodies”
resulting in visible
clumping or agglutination
of latex particles

21. Coagulase Test
Fibrin Fibrinogen
Tube Test: inoculate into tube of citrated plasma
Staphylococcus Aureus +
Other staphylococcus Strands -

22. Identification of
Streptococcus &
enterococcus Species

23. Blood Agar & Antibiotic Susceptibility
Antibiotics: Bacitracin & SXT
Differential Media
Exotoxin (Hemolysis)
Beta Beta
Alpha
Alpha

24. Bile Esculin (BEA)
Selective (Bile Salts)… Gram Negative MacConkey
Differential (Esculin & Ferric ammonia Citrate)
Hydrolyze the carbohydrate Esculin
Esculin
EsculetinGlucose
Ferric Ammonia Citrate
“form black precipitate”
Reacts
with
Esculin
(+) hydrolyzed to glucose & esuletin

25. Mannitol Salt Agar
Selective (7.5% NaCl) halotolerant….
Enterococcus + & Streptococcus -
Differential (Mannitol)
pH indicator:
phenol Red
Red  Yellow (Enterococcus Faecalis)

26. Rapid Strep TestSand which immunoassay: Test cassette contains a strip coated with
strep A antibodies
Strep A antigen
extracted from
throat swab
will bind to the
antibodies
the antigen antibody complex
then binds to a second antibody
that is conjugated with a
chromogen “colored line”
* Wrong 15% of the time

27. Identification of
Gram Negative

29. MacConkey Agar
Selective:Crystal Violet
& Bile salts *Inhibit Gram positive &
Select Gram Negative
Differential:
Ferment Lactose?
1) fermented lactose lower pH of media
below 6.8 pinkish-Red
2) Did not ferment lactose “colorless”
Carbohydrate
Substrate Lactose
pH indicator
Neutral Red

30. Citrate UtilizationSelective Only (Sodium Citrate, Sodium & Water)
Enzyme:
Citrase
pH indicator:
Bromothymol Blue
CO2
Sodium
citrate
Oxaloactetic
acid
Acetic
acid
pyruvate
Alkaline
Sodium
bicarbonate
pH
Indicator
Reacts
with
Na+ & H2O
Reacts
with

31. Urease Test
Enzyme:
Urease
Substrate:
Urea
pH Indicator
Phenol Red
urea
ammonia carbon dioxide
& water
Phenol
red
Reacts
with

32. Nitrate ReductionDifferential Only (Beef extract, Peptones & Nitrate)
Enzyme:
Nitrate Reductase
Anaerobically reduce nitrate  nitrite  Ammonia or completely to Nitrogen
(5 drops of Each)
Nitrate A = Sulfanilic Acid
Nitrate B = Alpha-naphythylamine
Yellow Positive
(nitrates reduced beyond
nitrites to ammonia or
molecular nitrogen)
RED Positive
reduced Nitrate  Nitrite
Yellow Add Zinc
RED Negative
(The zinc reduced
the nitrate NOT the
bacteria)

33. MRVP Test
Methyl Red (MR) Test Vogues- Proskaur (VP) Test
Ferment glucose & produce more than
one acid end product Lactic, Acidic
and formic acids
STRONG enough to overcome
phosphate buffering system
Positive
Reaction:
Red pH of 4.4 or lower
Ferment glucose but only produce ONE acid
end product “usually acetic acid”
The acid produced initially lowers the pH of
the media but is quickly converted to
acetylmethylcarbinol which leads to a pH of
6.2. Reagents react with acetylmethycarbinol
Positive
Reaction:
Red Band at the top which will diffuse
over time
Never can be
Both!!!!!
*Can be both
Negative

34. SIM agar TestContains: Amino acid Tryptophan
(peptones, sodium thiosulfate & ferrous sulfate)
Indole Production:
Tryptophanase degrade tryptophan into indole, pyruvate and ammonia.
*kovac’s Reagent contains HCL, butanol, and para-dimethyl-
amino-benzaldehyde (pdaba).
Hydrogen Sulfide Production
Thiosulfate reductase can reduce sulfur to hydrogen sulfide gas
Motility:
Motile vs. Nonmotile
“Sodium
thiosulfate”
Hydrogen sulfate
Ferrous sulfate
Reacts
with
Tryptophan
Indole
Pyruvate ammonia
acidified butanol
extracts any indole that is
produced & bring to
surface
pdaba
Reacts
with

35. Lysine Iron Agar
Deamination Aerobic (slant)
Deaminate positive
Deaminate Negative
Decarboxylation Anaerobic (Butt)
Decarboxylation Positive
Decarboxylation Negative
Hydrogen Sulfide
Thiosulfate reductase can reduce sulfur to hydrogen sulfide gas
0.1% Lysine, Glucose, Peptones, Bromcresol purple, Sodium thiosulfate, ferric ammonia citrate
ammonia ferric ammonium citrate α-ketocarboxylic acid
Reacts
with
Cadaverine “diamine end”
Raise ph above 6.8
Bromcresol purpleCleaves carboxyl group from lysine
Reacts
with

36. Triple sugar Iron (TSI) Agar Test0.1% glucose, 1.0% sucrose , 1.0% lactose, Peptones, Phenol red, Sodium thiosulfate, Ferrous sulfate
Carbohydrates Substrates (Slant & Butt)
0.1% glucose, 1.0% sucrose, 1.0% lactose
*Fermentation of these carbohydrates into acid end products will result in
a yellow color (A) on the slant and/or Butt as the acid reacts with the
Phenol Red
*No fermentation results in a pink color (Alk)
Gas
Positive or Negative “look for cracks”
Hydrogen Sulfide
Thiosulfate reductase can reduce sulfur to hydrogen sulfide gas

37. Organism Catalase MSA
fermentation
DNase Novobiocin
Sensitivity
Staphylococcus aureus
+ + + S
Staphylococcus epidermidis
+ - - S
Staphylococcus saprophyticus
+ + - R
Staphylococcus Xylosus
+ +/-
Weak
+ R

38. Organism Catalase Hemolysis Bile
Esculin
SXT Bacitracin MSA
growth
Enterococcus
Faecalis
- α + S R +
Streptococcus
Bovis
- α + R R -
Streptococcus
mutans
- ϒ + S S -
Streptococcus
pyogenes
- β - R S -
Streptococcus
zooepidemicus
- β - S*
(usually 21mm
Barely)
R -

39. Triple Sugar Agar SIM deep Lysine Iron Agar
Gram Negative
Bacterium
Lactation
Fermentation
Slant
Butt
Gas
H2S
Urease
Citrate
Indole
H2S
Motility*
MR
VP
Nitrate
SlantLysine
deamination
ButtLysine
Decarboxylati
on
H2S*
Aeromonas
Hydrophila
- Alk A - - - + + - - + + - -
Alcaligenes
faecalis
- Alk Alk - - - + - - - - - - +
Citrobacter
freundii
+ A A + + - + - + + - + - -
Edwardsiella tarda - Alk A + + - - + + + - + - +
Enterobacter
aerogenes
+ A A + - - + - - - + + - +
Enterobacter
Cloacae
+ A A + - - + - - - + + - -
Escherichia coli + A A + - - - + - + - + - -
Morganella
morganii
- Alk A + - + + + Weak
+
+ - + - -
Proteus mirabilis - Alk A - + + + - + + - + + -
Proteus vulgaris - A A - + + + + + + - + - -
Providencia
alcalifaciens
- Alk A - - - + - - + - + + -
Providencia
rettgeri
- Alk A - - + + + - + - + + -

40. Appendix A
Day 1
Gram + (Coccus)
Gram – (Rod)

41. Nutrient Agar Plate Not selective
 Not differential

42. C-CNA Agar PlateSelective: Colistin + Nalidixic Acid
Gram Positive
Differential: Blood agar
Beta, Alpha, Gamma

43. MacConkey Agar Selective: Crystal Violet & Bile Salts
Gram Negative
 Differential: Substrate Lactose & pH
indicator “Neutral Red”
RED  Pink

44. Gram Stain

45. Appendix A
Day 2

46. Alcaligenes Faecalis
Alk
Alk
TSI agar
No Gas
No H2S
Urease
Negative
Citrate agar
Positive
SIM agar
Indole
Negative
& No H2S
MR
Negative
VP
Negative
Nitrate
Reductase
Negative
(turned
when zinc
added)
Lysine
Deamination
Negative
Lysine
Decarboxylation
Positive

47. Staphylococcus
saprophyticus
Catalase: Positive

48. Yogurt, Food
& Water
microbiology

49. Yogurt
MicrobiologyHeat to boiling milk
Cool to 55ºC
Lactobacillus bulgaricus (Acidophile)
Streptococcus thermophiles (Thermophile)
Incubate @ 45ºC
Biochemistry: Fermentation (lactose)  lactic acid (Lower pH)
Cow’s milk pH: 7
calcium caseinate (colloidal suspension)
calcium lactate free (soluble) casein

50. Food Microbiology
Salmonella & Shigella
Selective: Bile salts
(select gram negative enteric bacteria)
Differentiate
Carbohydrate:
Lactose fermenters
pH: Neutral Red
H2S: ferric citrate
Escherichia coli
Salmonella
shigella
Chicken Beef

51. Water MicrobiologyMPN “Most probable number”
Tube inside “Durham tube”
Carbohydrate: lactose broth
What is the pH indicator: Phenol Red
Small inverted tube present in the MPN tubes: Durham tubes
Presumptive Test
10ml 1ml 0.1ml
Sewage Water 3/3/3 = >1000 coliforms/ 100ml of water sample
Fecal coliforms
“Indicator organisms” water has
been contaminated with human or
animal feces
Obviously
negative if
PINK!!
Gas
Positive: Yellow
(Lactose fermentation)
+ Gas production
(Floating Durham)
Negative: Pink or
doesn’t float

52. ELISA testing &
Epidemiology

53. EPIDEMIOLOGY
InvestigationEpidemiologist Goals:
1.) Case of disease
2.) Route of Transmission
3.) Trace patter to identify original carrier “patient 0”
Test Tubes  “Body Fluids”
Phenol red  infected??
Who
infected
me??

54. ELISA testingTesting Sample A & B
Before Antibodies are added:
(AG) Purified Antigen
Antibodies added:
Positive Control
Negative Control
Sample A
Sample B
After Antibodies are added:
(SA) Secondary Antibody  Attach to the first antibody & has a chromogen attached
(SUB) Enzyme substrate  detection reagent
(-) (-) (-) (+) (+) (+) (A) (A) (A) (B) (B) (B )