microbiology practical 2!!!! i will miss this class! (ilana kovach)
TRANSCRIPT
Microbiology 2420Practical 2
Ilana Kovach
Identification of Bacillus Species
Starch media Substrate:Polysaccharide Starch
Enzyme:Amylase & Maltase
starch
+
+
-
-Maltose (disaccharide)
Glucose (monosaccharide)
Amylase Maltase
Lipid mediaSubstrate:Tributyrin
Enzyme:Lipase
Triglycerides
+
+ (Slightly)
+
-
Glycerol
Fatty acids
Caesin MediaSubstrate:Caesin
Enzyme:Protease & Casease
Casein
++
+ -
Peptones Polypeptides dipeptidesAmino
acids
+
++ - -
-
Gelatin mediaSubstrate:Gelatin (Protein produced by hydrolysis of Collagen)
Enzyme:Gelatinase
Gelatin
+ -
Amino acid components “cause gelatin to liquefy”
Gelatinase
Mannitol Salt agarSelective (7.5% NaCl) halotolerant
Differential (Mannitol)
pH indicator:
phenol Red Red Yellow
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 NitriteYellow Add Zinc
RED Negative
(The zinc reduced
the nitrate NOT the
bacteria)
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
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
Transformation
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)
TransformationLB Before: Growth
LB after: Growth
LBamp Before: No growth
“NOT resistant to ampicillin”
LBamp After: Growth
“Resistant to ampicillin” &
Glows
identification of StaphylococcusSpecies
Catalase TestH2O2 (Hydrogen Peroxide) 2H20 (Water) + O2 (Oxygen)
Staphylococcus +
Streptococcus/
Enterococcus -
Mannitol Salt Agar Selective (7.5% NaCl) halotolerant
Differential (Mannitol)
pH indicator:
phenol Red Red Yellow
+
+weak
+
-
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
+ +
- -
Mueller Hinton Agar Test Susceptibility of Novobiocin
>18mm
Nutrient agar (Neither
selective or differential) Controlled: Levels of thymine, Thymidine, Ca+ ions & Mg
+
+-
-
Rapid Staph TestLatex 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
Coagulase TestFibrin Fibrinogen Tube Test: inoculate into tube of citrated plasma
Staphylococcus Aureus +
Other staphylococcus Strands -
Identification of Streptococcus & enterococcus Species
Blood Agar & Antibiotic Susceptibility
Antibiotics: Bacitracin & SXT
Differential Media
Exotoxin (Hemolysis)
Beta Beta
AlphaAlpha
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
Mannitol Salt Agar Selective (7.5% NaCl) halotolerant….
Enterococcus + & Streptococcus -
Differential (Mannitol)
pH indicator:
phenol Red Red Yellow (Enterococcus Faecalis)
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
Identification of Gram Negative
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”
CarbohydrateSubstrate Lactose
pH indicator Neutral Red
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
Urease Test
Enzyme:Urease
Substrate:Urea
pH Indicator Phenol Red
urea
ammonia carbon dioxide
& water
Phenol
red
Reacts
with
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 NitriteYellow Add Zinc
RED Negative
(The zinc reduced
the nitrate NOT the
bacteria)
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
SIM agar Test Contains: 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
Lysine Iron AgarDeamination Aerobic (slant)
Deaminate positive
Deaminate Negative
Decarboxylation Anaerobic (Butt)
Decarboxylation Positive
Decarboxylation Negative
Hydrogen SulfideThiosulfate 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
Triple sugar Iron (TSI) Agar Test 0.1% 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)
GasPositive or Negative “look for cracks”
Hydrogen SulfideThiosulfate reductase can reduce sulfur to hydrogen sulfide gas
Organism Catalase MSA
fermentation
DNase Novobiocin
Sensitivity
Staphylococcus aureus + + + S
Staphylococcus epidermidis + - - S
Staphylococcus saprophyticus + + - R
Staphylococcus Xylosus + +/-
Weak
+ R
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 -
Triple Sugar Agar SIM deep Lysine Iron Agar
Gram Negative
Bacterium
Lact
ati
on
Fer
men
tati
on
Sla
nt
Bu
tt
Gas
H2S
Ure
ase
Cit
rate
Ind
ole
H2S
Moti
lity
*
MR
VP
Nit
rate
Sla
nt
Lysi
ne
dea
min
ati
on
Bu
tt L
ysi
ne
Dec
arb
oxyla
ti
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 - - + + + - + - + + -
Appendix A Day 1Gram + (Coccus)Gram – (Rod)
Nutrient Agar Plate Not selective
Not differential
C-CNA Agar PlateSelective: Colistin + Nalidixic Acid
Gram Positive
Differential: Blood agar
Beta, Alpha, Gamma
MacConkey Agar Selective: Crystal Violet & Bile Salts
Gram Negative
Differential: Substrate Lactose & pH
indicator “Neutral Red”
RED Pink
Gram Stain
Appendix A Day 2
Alcaligenes Faecalis
Alk
Alk
TSI agarNo GasNo H2S
UreaseNegative
Citrate agarPositive
SIM agarIndole Negative& No H2S
MRNegative
VPNegative
Nitrate ReductaseNegative (turned when zinc added)
Lysine DeaminationNegative
LysineDecarboxylation
Positive
Staphylococcus
saprophyticusCatalase: Positive
Yogurt, Food
& Water
microbiology
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
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
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
ELISA testing &
Epidemiology
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??
ELISA testing Testing 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 )