Download Gram negative rods and anaerobes

GRAM NEGATIVE RODS: ENTEROBACTERIACEAE
Facultative anaerobes, found in the GI tract
LACTOSE +: Coliforms, mainly non-pathogenic. Studied because their
presence is an easy way to detect water contamination.
Includes: E. coli, Enterobacter, Citrobacter, Klebsiella.
MEDIA: EMB and MacKonkey’s (MAC)
LACTOSE -: Shigella, Salmonella. Proteus, Providencia, Serratia.
Shigella: Effective dose = 200 cells. H2S negative.
Salmonella: H2S positive, acid resistant, normal flora in chicken, Typhi is not
as common, and heads for liver and gall bladder;99% identical to Escherichia.
Proteus: Fairly common UG pathogen (the most common is E. coli)
Providencia and Serratia: Mainly seen in immunocompromised.
MEDIA: Hektoen or Salmonella/Shigella media. Both medias have dyes, acid
indicators, and also bile to inhibit gram +.
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ENTEROBACTERIA LAB TESTS:
Urease media:
Has phenol red pH indicator: Base  pink (+)
Eosin Methylene Blue agar (EMB)
Differential because of lactose
Selective because of two dyes (eosin and Methylene blue)
It allows only gram – to grow because the gram + thick cell wall
inhibits the dyes.
Has pH indicators: Acid pink (+) Colorless is negative.
E. coli looks shiney metallic green on EMB.
MacConkey’s Media
Selective because it has Crystal Violet (inhibitory of Gram +)
and because it has bile acids (eventually causes cloudy ppt)
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Phenol Red Tubes (PR Tubes): contain glucose, sucrose, and/or lactose
Is a pH indicator (0=acid:yellow, 7=neutral:red, 14=base:pink)
If organism can ferment sugar acid = yellow
Duram tube is inverted inside PR tube, catches H+ and CO2 gases.
Record results: K=red, A=yellow, G=gas
Kligler’s Iron Agar (KIA): Slants with reddish media
Has two fermentable carbohydrates: lactose (10x), glucose (1x)
Stab the butt, then streak the slant:
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Record slant/butt by color:
Non-fermenter= red= K/K
Darker red at surface= Alk/Alk
Ferment glucose but not lactose: K/A
Whole tube will be yellow in 12 hours
By 24 hours, surface=red, butt=yellow
Ferments both sugars: A/A
Gases will cause cracks in media: A/A G
Cystine is an amino acid with sulfur; the bacteria want to cleave the
sulfur and make it H2S gas, so it can eat the sugar.
Stab will turn black if the iron was reduced: Fe + H2S  FeS (black)
NOTE: To record “all yellow with gas and iron use” = A/A +G, H2S +
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Triple Sugar Iron Agar (TSI)
glucose 1x
lactose 10x
sucrose 10x
No sugars fermented: K/K
Glucose only fermented: K/A
Either lactose or sucrose fermented: A/A
Sulfide Indole Motility (SIM) Media (semi-solid media)
Sulfide: Cysteine + Iron  Black (+)
Indole: side group of triptophan amino acid: indicates presence of triptophase
TRP  Indole (add Kovak’s reagent)  red (+)
Use straight stab with needle to check for motility. If stab is black = sulfide.
NOTE: Salmonella and Proteus are SIM black
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IMViC media: Positive = red-red-red-blue
Indole  inoculate TRP
Methyl Red  MRVP tubes, add M.R.
Voges Proskavar  MRVP tubes to check for acetoin production
Citrate: green slants = Simmons Citrate. Inoculate slant only.
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ORGANISM
E. coli
EMB
Shiney
Green
K. pneumoniae Pink
C. divers.
Pink
E. aerogenes
Pink
C. freundii
pink
Salmonella
Proteus
MAC G
L
S
C
U SIM
Pink A/G A/G A/G -
-
-
Pink
Pink
Pink
Pink
+
+
+/-
+/+/+
+
A/G
A/G
A/G
A/G
A/G
A/G
A/G
A/G
A/G
A/G
A/G
A/G
+
+
+
+
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NITRATE TUBES
NO3 (Nitrate)  NO2 (Nitrite)    N2 (Nitrogen gas)
ADD: Reagent A = sulfamilic acid
Reagent B = dimethyl α napthylamide
If it turns red = Nitrate = +
If no color change, it’s either negative, or nitrate was all used up.
So, ADD Reagent C = zinc
A + B = red = +
A + B = no change = add reagent C
A + B + C = no change = nitrate was used up = +
A + B + C = red = nitrate untouched = -10
HEKTOIN (green plates)
SALM/SHIG (S/S = red plates)
Bile
Dyes
H2S
lactose + = yellow-orange
lactose - = blue/green
sucrose
Bile
Dyes
H2S
lactose + = pink
lactose - = colorless
no sucrose
NOTE:
Salmonella has “Bulls-eye colonies” = blue/green colonies with black center.
Phenylalanine Slants (Phe slants) have amino acid in them. We’re looking for
phenylalaminase. Just streak slant, incubate, then add FeCl3 (Ferric
Chloride). If it can deaminate phenyalanime, it will become phenyl pyrate and
turn green = + (all the genera that start with a “P” are positive for Phe
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slants)
Includes Providencia and Proteus.
NOTE: Cysteine is found in any media that has “I”. Black is +
TEST FOR DECARBOXYLATION OF LYS, ARG, ORN
Purple tubes have pH indicator, plus a base media of glucose.
Cover with mineral oil, since O2 interferes with action of enzymes.
In 12 hours, the presence of deCO2ase will turn media yellow, but its pH will
turn it back to purple in 48 hours. PURPLE IS + and YELLOW IS If they don’t decarboxylase the amino acid, tube will be yellow in 48 hours.
The base turns yellow because glucose is fermented to an acid.
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Which amino acid in above picture was decarboxylated? (ARG)
LIA = LYSINE IRON AGAR: purple slants (stab and streak).
Purple=K/K: This test is positive (e.g. E. coli)
Yellow =A/A: This test means glucose and lysine are fermented, but not
decarboxylated, so test is negative.
Butt is Purple: Positive for decarboxylase.
Stab is black: H2S positive
Top is Red: Positive for Deaminase
POSSIBLE COMBINATIONS: K/A R/A K/K K/A H2S+
K/K H2S+
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DILUTION SERIES
TDF (Tube Dilution Factor): Amount of dilution that occurs in any tube
during the whole process:
Sample volume
Sample volume + Tube volume
SDF (Serial Dilution Factor): Multiply the TDF’s: 0.1 x 0.1 x 0.1 = 10 -3
PDF (Plate Dilution Factor): The fraction that was put on the plate:
Volume Plated
1 ml
FDF (Final Dilution Factor): FDF = PDF x SDF
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When there are four plates with various numbers of colonies, choose the ones
with colony counts of 30-300, and average them.
Colony Forming Units = CFU
CFU = 31__
FDF
10 -4
= 310,000
Average = 340,000
375__
10 -3
= 375,000
Dilutions give viable cell counts. They are accurate if pipetting is accurate.
Each CFU may represent more than one cell.
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Hemacytometer
1cm = 10mm
10mm x 10mm x 10mm = 1 ml
1000mm3 = 1ml
1mm3 = 1 µl
The Hemacytometer holds a cover slip 1mm above it = 0.1µl
= 1 x 1 x 0.1 = 10 -4 ml
Count any four squares and take the average. (e.g. average = 30)
Multiply by 16 or 25 (the number of blocks per grid) (e.g. 30 x 16 = 4800)
Divide by the volume over the big square: 10 -4
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SUMMARY OF HEMACYTOMETER CALCULATIONS
1. Count four squares and average
2. Average x 16 (or 25) grids
= bacteria per square
3. Bacteria per square
10 -4
= bacterial per ml
4. Bacteria per ml x 10-fold dilution = bacteria per ml estimate
5. Want =
Have
? ________ ?
per ml estimate
= ml of culture needed to add to
water to make 1 ml
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Immunofluorescence
Benefits:
Direct detection of bacteria
Specificity
Direct Immunofluorescence: 1° antibody (mouse) is bound to bacteria, and is
also conjugated to fluorescent dye.
Indirect Immunofluorescence (e.g. ELIZA):
2° antibody (goat anti-mouse) is conjugated to the 1° antibody (mouse)
Benefits:
Get amplification to signal (it looks brighter, easier to see)
Cheaper (only need one conjugated antibody)
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INDIRECT IMMUNOFLUORESCENCE PROCEDURE
(of Shigella somnei)
Use Phosphate Buffered Saline (PBS)
1. Add 1 ml PBS to microfuge tube
2. Suspend organism to light turbidity
3. Spin (w min @ 12,000 rpm), keep pellet only
4. Add 1 ml PBS
5. Spin, keep pellet
6. Repeat steps 4, 5
7. Add 1 ml PBS and 2 µl 1° antibody
8. Incubate 37° C x 2 hours
9. Spin, keep pellet
10. Add 1 ml PBS
11. Repeat steps 9, 10
12. Add one ml PBS and 2 µl 2° antibody (fluorescene-labled goat anti-mouse)
13. Incubate at room temp for two days
14. Spin, keep pellet
15. Add 1 ml PBS
16. Repeat 14, 15
17. Put 10 ml of final suspension on slide, add cover slip and immersion oil
18. Observe with phase contrast
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ANAEROBES
The spectrum of O2 tension:
Amospheric
Obligate Aerobes (humans, pseudomonas)
Microaerophiles (atmospheric is too much)
O2 tension
Facultative Anaerobes (Can use it if it’s there; if not, it uses
anaerobic respiration or fermentation, e.g. E. coli)
Aerotolerant (don’t use it, but tolerate it; Streptococci)
Anaerobic
Obligate Anaerobes (inhibited, bacteriostatic by O2)
Strict Anaerobes (even a small amount of O2 kills it)
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ANAEROBIC JAR: Contains atmospheric O2.
Add GasPak chemicals (cut the corner, add 10 ml water(
Sodium borohydride  H2
Citric Acid  acidic  CO2
Sodium Bicarbonate  CO2
Palladium pelletd  catalyst to make H and O2 combine to H2O
Add pH indicator strip: Methylene Blue (a simple stain, starts out white)
If O2 is present, strip turns blue-green.
If no O2 present, strip stays white/clear
This test strip has three things:
1) pH indicator
2) O2 indicator
3) simple stain
Another test strip is called Resazurin. It turns pink with O2, clear without.
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Toxic metabolic end products of O2 determine the bacteria’s sensitivity to O2.
O2  H2O2 (superoxide anion), which denatures proteins and enzymes, and
damages the DNA, etc.
Humans make catalse and SOD, which destroys the above toxic end products.
Anaerobes don’t make SOD or catalase, so they die with the presence of O2.
Gram + rods
Clostridium perfringens (makes proteases and spreading factors) gas gangrene.
C. tetani  tetanus
neurotoxins
C. botulinum  botulism
Terminal endospores: C. tetani
Medial endospores: C. botulinum and perfringens
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Gram + cocci: Peptostreptococcus
Gram neg rods: Bacteroides (GI), and Fusobacterium (mouth).
Anaerobic Media and Tests
BRU/BA = Blood Agar
PEA = Phenyl ethylamine
BBE = Bacteroides Bile Esculin (for anaerobes: Black is +)
EYA + Egg Yolk Agar (a lipid plate, to detect lipases)
Catalse (most are negative)
Indole test: Positive = blue
Motility
Nitrate Utilization: Positive = red
Oxidase: Positive = dark blue (rare)
K = Kanamycin disc
C = Colistin disc
V = Vancomycin disc
SPS = Sodium Polyanethol Sulfonate
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SIX ORGANISMS TO KNOW
1. Fusobacterium nucleatum: Gram – rods with pointed ends
5. Bacteriodes fragilis: BBE +
12. Clostridium difficile: Hazy hemolysis on BA, Gram +
17. Clostridium perfringes: double-zone hemolysis, Gram +
46. Bacteriodes uralyticus: Gram – rods, pitting, Urease + (pink)
22. Peptostreptococcus: The only Gram + cocci
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