Download Clinical Bacteriology II (MLAB 481)

Clinical Bacteriology II
(MLAB 481)
Supervisor
Mohammed A. Alissa
Student Name:
Student ID:
Table of Contents Safety Precautions in the Microbiology Laboratory and Basic Bacteriology
Techniques .................................................................................................................................. 1 Enterobacteriaceae .................................................................................................................... 3 Culture of Enterobacteriaceae (Non-lactose Fermenters ‘NLF’) ................................... 8 Biochemical Reactions of Enterobacteriaceae ................................................................. 13 API 20E ..................................................................................................................................... 37 Pseudomonas aeruginosa ....................................................................................................... 40 Haemophilus spp. .................................................................................................................... 44 Campylobacter spp. ................................................................................................................. 48 Vibrio spp. ................................................................................................................................. 52 Clostridia .................................................................................................................................. 56 Bacteroides spp. ....................................................................................................................... 61 Safety Precautions in the Microbiology Laboratory and
Basic Bacteriology Techniques
A. Lab Safety
Bacteria and viruses are infectious in the lab as well as in the community
and care must be taken at all times to avoid infecting either yourself or
others working with you.
There are three main routes by which microbes can enter the body:
i. Respiratory: by breathing in infectious aerosols*
ii. Alimentary: by swallowing infectious material.
iii. Dermal: through the skin via recognizable cuts or minute abrasions.
* Aerosols are minute, bacteria-laden, particles liberated into the
atmosphere during many common lab manipulations, e.g. opening culture
bottles, flaming wire loops and preparations of smears for staining. Small
particles (<10 µm diameter) remain suspended in the atmosphere and may
enter the body during respiration. Large particles will eventually settle on
the worktops and surrounding objects.
Use of some reagents also carries certain hazards. All student practical
classes are subject to COSHH (Control of Substances Hazardous to Health)
regulations.
B. Safety Rules
1. ALWAYS WEAR A HOWIE-STYLE WHITE COAT in the lab.
Always keep it buttoned up. This keeps your outdoor clothing free from
contamination.
1
2. Wear gloves for handling bacterial cultures and carrying out staining
procedures. Please tell a demonstrator if you have an allergy as alternative
gloves are available on request.
3. Dispose of waste from the practical sessions in the yellow clinical waste
bags provide, not the black ‘household’ waste bins. Loops and pipettes
should be placed in the beakers of disinfectant and glass slides in sharps
bins.
4. Tie back long hair.
5. Do not put personal belongings on the working benches.
6. Do not sit on benches.
7. Never eat, drink or even bring food into the lab.
8. Protect cuts and scratches with a suitable waterproof plaster while
working.
9. Do not lay loops, pipettes or spreaders on the bench tops. Discard directly
and immediately into disinfectant solution.
10. ALWAYS WASH YOUR HANDS before leaving the lab.
11. ACCIDENTS OF ANY KIND, whether breakage, spillage (however
minor) or personal injury, must be reported immediately to the demonstrator
or lab supervisor.
12. Read and observe COSHH notes within the manual.
I hereby attest that I have read and understand the information provided to
me regarding the safety rules.
Name:
Signature:
2
Enterobacteriaceae
- General characters:
1. Gram negative, rods.
2. Facultative anaerobes.
3. Ferment glucose (produce acid or acid + gas).
4. Oxidase negative.
5. Reduce nitrate (NO3) into nitrite (NO2).
6. Motile by peritrichous flagella and non-motile organisms.
7. Non-spore formers.
Classification of Enterobacteriaceae based on lactose fermentation
Lactose
fermenter
(LF)
Non-lactose
fermenter
(NLF)
E.coli
Salmonella spp.
Klebsiella spp.
Shigella spp.
(except Shigella
sonii which is
late lactose
fermenter 'LLF')
Citrobacter spp.
Proteus spp.
(except proteus
mirabilis which
is LLF)
Enterobacter
spp.
Yersinia spp.
3
Media
Lactose fermenter
Non-lactose
(LF)
fermenter
Indicator
(NLF)
MacConkey agar
Pink colonies
Colorless
Neutral red
Pink colonies
Colorless
Methylene Blue
Pink colonies
Colorless
Neutral red
Yellow colonies
Colorless
Phenol red
Orange colonies
Colorless
Thymol blue +
(red)
Eosin Methylene
Blue (EMB)
E.coli give metallic
(reddish-brown)
sheen
Deoxycholate
Citrate agar
(DCA) (pink)
Xylose Lysine
Deoxycholate agar
(XLD) (deep red)
Hektoen Enteric
agar (HE) (olive)
Acid fuchsin
Cystine Lactose
Yellow colonies
Colorless
Electrolyte
Deficient (CLED)
(sky-blue)
4
Bromothymol blue
MacConkey agar and EMB are primary (enriched) media, selective media
for enteric Gram negative rods, as well as differential media for lactose and
non-lactose fermenters.
XLD, DCA & HE are selective media used in the isolation of Salmonella
spp. and Shigella spp. from stool samples, as well as differential media for
lactose and non-lactose fermenters.
CLED is primary medium is used for isolation of pathogens from urine
samples, as well as differential media for lactose and non-lactose fermenters.
-Describing the colonies on the media
Large, smooth, (LF) or (NLF), (color) or (colorless) colonies.
Klebsiella spp.à large, smooth, LF, mucoid, (color) colonies.
5
E. coli on EMB:
Large, smooth, LF, greenish black circular colonies with metallic
appearance under the light (metallic sheen).
6
Exercise:
1) Based on the bacterium that you have, do the following:
a) Gram stain:
………………………………………………………………………………...
b) Culture the bacterium on the media that you have, mention the name
of these media and describe the colonies on them:
………………………………………………………………………………...
7
Culture of Enterobacteriaceae (Non-lactose Fermenters
‘NLF’)
- Organisms: Salmonella spp., Shigella spp., Proteus spp.
- Salmonella spp. and Shigella spp. on MacConkey agar, EMB, and
CLED are:
Large, smooth, NLF, colorless colonies.
- Salmonella spp. on XLD, DCA, and HE is:
Large, smooth, NLF, colorless colonies may be with black center.
8
- Shigella spp. on XLD, DCA, and HE is:
Large, smooth, NLF, colorless colonies without black center.
Selenite F broth: enrichment medium for Salmonella spp. and Shigella spp.
Turbid
Clear
There is growth
There is no growth
*Note: Salmonella spp. is motile while Shigella spp. is non-motile.
9
- Proteus spp. produces swarming on blood agar and nutrient agar (which is
waves starting from the inoculation point) + fishy smell.
- Proteus spp. needs salt in order to produce swarming. Because of this,
it cannot produce swarming on CLED which is electrolyte (salt)
deficient.
- Proteus spp. on CLED à Large, smooth, NLF, colorless colonies
without swarming because the medium is salt free.
10
- Proteus spp. on XLD, DCA, and HE is:
Large, smooth, NLF, colorless colonies with black center.
* Note: Proteus spp. is considered as normal flora in the gut.
11
Exercise:
1) Based on the bacterium that you have, do the following:
a) Gram stain:
………………………………………………………………………………...
b) Culture the bacterium on the media that you have, mention the name
of these media and describe the colonies on them:
………………………………………………………………………………...
12
Biochemical Reactions of Enterobacteriaceae
1. Indole Test (I)
- The indole test determines the ability of bacteria to spilt tryptophan into
indole, pyruvate and ammonium.
- Substrate: tryptophan.
- Enzyme: tryptophanase.
- Procedure:
a) Inoculate the organism in the tryptophan broth.
b) Incubate the inoculated broth at 37°C for 24 hours.
c) Few drops of Kovac’s reagent will be added to the medium in the next
day.
d) Read the result:
Indole Test
(I)
Red ring
(positive)
Yellow ring
(negative)
E. coli
Klebsiella
spp. except
K. oxytoca
13
2. Methyl Red Test (MR)
- MR test is used to determine the ability of an organism to produce mixed
acids by fermentation of glucose.
- Explanation: MR test is used to identify enteric bacteria based on their
pattern of glucose metabolism. All enterics initially produce pyruvic acid
from glucose metabolism. Some enterics subsequently use the mixed acid
pathway to metabolize pyruvic acid to other acids, such as lactate, acetate,
etc. These bacteria are called MR positive i.e. E. coli. Other enterics
subsequently use the butylene glycol pathway to metabolize pyruvic acid to
neutral end products. These bacteria are called MR negative i.e. Klebsiella
spp.
- Substrate: glucose.
- Methyl red reagent is an indicator dye that turns red in acidic solutions (PH
4.4).
14
- Procedure:
a) Inoculate the organism in glucose phosphate peptone water.
b) Incubate the inoculated broth at 37°C for 24 hours.
c) Few drops of methyl red reagent will be added to the medium in the next
day.
d) Read the result:
Methyl
Red Test
(MR)
Red
(positive)
Yellow
(negative)
E. coli
Klebsiella
spp.
15
3. Voges Proskauer Test (VP)
- VP test is used to determine the ability of some organisms to produce a
neutral end product, acetyl methyl carbinol (acetoin) from glucose
fermentation. In the presence of atmospheric oxygen and 40% KOH, acetoin
is converted to diacetyl. α-Naphthol serves as catalyst to bring out red color
complex.
- Substrate: glucose.
-Procedure:
a) Inoculate the organism in glucose phosphate peptone water.
b) Incubate the inoculated broth at 37°C for 24 hours.
c) Few drops of KOH and α-Naphthol will be added to the medium in the
next day.
d) Read the result:
Voges
Proskauer Test
(VP)
Red (positive)
Yellow
(negative)
Klebsiella spp.
E. coli
16
4. Citrate Test (C)
The citrate test is used to distinguish between members of the
Enterobacteriaceae family based on their metabolic by-products. In the most
common formulation, citrate is the sole source of carbon in the Simmons
citrate medium while inorganic ammonium salt (NH4H2PO4) is the sole
fixed nitrogen source. When an organic acid such as citrate is used as a
carbon and energy source, alkaline carbonates and bicarbonates ultimately
are produced. The visible presence of growth on the medium and the change
in pH indicator color due to the increased pH are the signs that an organism
can import citrate and use it as a sole carbon and energy source; such
organisms are considered to be citrate positive.
Citrate, a Krebs cycle (i.e., TCA cycle or citric acid cycle) intermediate, is
generated by many bacteria; however, utilization of exogenous citrate
requires the presence of citrate transport proteins (permeases). Upon uptake
by the cell, citrate is cleaved by citrate lyase to oxaloacetate and acetate. The
oxaloacetate is then metabolized to pyruvate and CO2.
citrate = oxaloacetate + acetate
oxalacetate = pyruvate + CO2
17
Further metabolic breakdown is dependent upon the pH of the medium.
Under alkaline conditions, pyruvate is metabolized to acetate and formate.
pyruvate = acetate + formate
At pH 7.0 and below, lactate and acetoin are also produced.
Pyruvate = acetate + lactate + CO2
Pyruvate = acetoin + CO2
The CO2 that is released will subsequently react with water and the sodium
ion in the medium to produce sodium carbonate, an alkaline compound that
will raise the pH. In addition, ammonium hydroxide is produced when the
ammonium salts in the medium are used as the sole nitrogen source. The
bromothymol blue pH indicator is green at neutral pH. With an increase
in medium pH to above 7.6, bromothymol blue changes to blue.
Citrate Test
(c)
Blue (positive)
Green
(negative)
Klebsiella spp.
E. coli
18
E. coli
Klebsiella
I
M
Vi
C
+
+
-
-
- (except K.
oxytoca)
-
+
+
19
5. Urea Test
- Indicator: phenol red.
Urease Test
Red (positive)
Yellow
(negative)
Proteus
Salmonella
20
6. Triple Sugar Iron (TSI) (reddish-orange color)
Slant
Butt
Butt
- TSI is a tubed differential medium used in determining carbohydrate
fermentation and H2S production.
- TSI contains 3 types of sugar: 0.1% glucose (in the butt), 1% lactose and
1% sucrose (in the slant).
- Indicator: phenol red.
- The slant is aerobic, while the butt is anaerobic.
- Based on carbohydrate utilization and hydrogen sulfide production, a
TSI slant can be interpreted in several ways:
a) Glucose fermenter (A) or non-glucose fermenter (NC or K)
- The bacterium is called glucose fermenter when the butt is changed to
yellow due to acid production (A).
- The bacterium is called non-glucose fermenter when the butt is not
changed (NC), or changed to red (K) due to utilization of peptones
anaerobically which causes the release of ammonia (NH3) resulting in
21
elevating the pH (alkaline) making the pH indicator, phenol red, turning
from pink to red.
- All Enterobacteriaceae are glucose fermenter.
b) LF (A) or NLF (NC or K)
- The bacterium is called lactose fermenter when the slant is changed to
yellow due to acid production (A).
- The bacterium is called non-lactose fermenter when the slant is not
changed (NC), or changed to red (K) due to utilization of peptones
aerobically which causes the release of ammonia (NH3) resulting in
elevating the pH (alkaline) making the pH indicator, phenol red, turning
from pink to red.
c) Gas production (g+) or no gas production (g-)
- Gas production (CO2 and O2) is detected by splitting of the agar. In some
cases, so much gas is produced that the agar is pushed to the top of the tube.
However, some strains do not produce gas.
d) H2S Production (H2S+) or no H2S Production (H2S-)
The black precipitate indicates that the bacteria were able to produce
hydrogen sulfide (H2S) from sodium thiosulfate. Because H2S is colorless,
ferric ammonium citrate is used as an indicator resulting in the formation of
insoluble ferrous sulfide. Formation of H2S requires an acidic environment;
even though a yellow butt cannot be seen because of the black precipitate,
the butt is acidic.
22
- How to write the result:
Slant (A or NC or K)/ butt (A or NC or K) g+ g- H2S+ or H2S-
Examples:
E. coli à A/A g+ H2S-
Klebsiella à A/A g+ H2S-
Salmonella à K/A g- H2S+
Proteus à K/A g- H2S+
Shigella à K/A g- H2S- (Shigella sonnei à A/A g- H2S-)
E. coli
Klebsiella
Salmonella
Proteus
Shigella
23
TSI Medium
7. Sulfur Indole Motility Media (SIM)
- S stands for H2S
- I stands for Indole Test (Kovac’s reagent will be added)
- M stands for motility (flagella ‘H antigen)
- Procedure:
a) Inoculate the organism in the gel medium.
b) Incubate the inoculated medium at 37°C for 24 hours.
c) Read the H2S and motility results
c) Few drops of Kovac’s reagent will be added to the medium for indole test.
d) Read the indole test result
*note: if black color seen in all the tube, this means the organism is motile.
SIM
S (H2S)
Black
(Positive)
Yellow
(Negative)
I (Indole
Test)
Red Ring
(Positive)
Yellow Ring
(Negative)
24
M (Motility)
Turbid
(Motile)
Clear (Nonmotile)
S
I
M
E. coli
-
+
+
Klebsiella
-
-
-
Shigella
-
-
Salmonella
+
-/+
(S. sonnei is
always indole
negative)
-
+
Proteus
+
- (P. vulgaris +)
+
Enterobacter
-
-
+
E. coli
Klebsiella
Shigella
Salmonella Enterobacter
Proteus
25
Proteus vulgaris
8. Oxidase Test
- The oxidase test is used to identify bacteria containing the respiratory
enzyme cytochrome c oxidase, which transfers electrons from cytochrome c
to oxygen.
- Positive result à purple color in 15 seconds. Color developing after a
long period should be ignored.
All Enterobacteriaceae are oxidase negative.
Positive 9. Catalase Test
- Catalase is an enzyme which catalyzes the dissociation of hydrogen
peroxide (H2O2) to oxygen and water.
2 H 2O2
Catalase
2 H 2O + O2
- Substrate: H2O2
- End product (positive result): gas bubbles (O2)
- Procedure: pick off a colony or group of colonies with a sterile loop and
smear onto a microscope slide, then add a drop of H2O2 with a Pasteur
pipette.
All Enterobacteriaceae are catalase positive.
26
Positive
10. Oxidation Fermentation Test (OF)
- OF test is used to determine whether the type of metabolism of the
bacterium is fermentative or oxidative.
Mineral
oil
27
Anaerobic
condition
Aerobic
condition
If the agar or broth turns yellow, acid is produced. If agar turns green, no
acid is produced.
- Oxidative metabolism may (O+/F-) or may not (O-/F-) cause an acid to be
produced for aerobic conditions. No acid will be produced for anaerobic
conditions.
Ex: Pseudomonas aeruginosa (O+/F-).
Ex: Micrococcus luteus (O-/F-).
- Fermentative metabolism will cause an acid to be produced for aerobic
and anaerobic conditions (O+/F+).
Ex: E. coli.
All Enterobacteriaceae are fermentative organisms.
11. Nitrate Reduction Test (Nitrate NO3 into nitrite NO2)
- Substrate: KNO3 or NaNO3
- Enzyme: nitrate reductase.
- Procedure:
a) Inoculate the bacteria in the nitrate reduction broth.
b) Incubate the inoculated broth at 37°C for 24 hours.
c) Nit 1 reagent (sulfanilic acid) and Nit 2 reagent (α-Naphthylamine) will
be added in the next day.
d) Read the result
- Red color: If the organism reduces nitrate into nitrite, Nit 1 & Nit 2 react
with nitrite and turn the color to red (Positive).
28
-Yellow color: Some organisms have strong enzyme which reduce nitrate to
N2 gas or ammonia. Therefore, Zinc dust (reducing agent) will be added to
show if the organism reduce nitrate into NO2 gas or the organism cannot
reduce nitrate. So, if yellow color is seen after adding the Zinc dust, this
means there is no nitrate in the broth and the organism has strong enzyme
(Positive). If red color is seen after adding the Zinc dust, this means the
organism cannot reduce nitrate and the Zinc dust reduce it (Negative).
All Enterobacteriaceae reduce nitrate into nitrite
Test
After adding NIT 1 &
NIT 2
After adding Zinc dust
Nitrate Reduction
Test
Red Color
(Positive)
Yellow Color
Yellow Color
(Positive) (the
organism has
strong Nitrate
reductase enzyme
29
Red Color
(Negative)
12. Decarboxylase Test
- Decarboxylase broth tests for the production of the enzyme decarboxylase,
which removes the carboxyl group from an amino acid.
- This test is useful for differentiating the Enterobacteriaceae.
- The three amino acids tested are arginine, lysine, and ornithine. Single
amino acid is tested in each tube. Each decarboxylase enzyme produced by
an organism is specific to the amino acid on which it acts. Therefore, we test
the ability of organisms to produce arginine decarboxylase, lysine
decarboxylase, and ornithine decarboxylase using three different but very
similar media.
30
- Indicator: bromocresol purple and cresol red.
- Enzyme: decarboxylase.
- Requirements of decarboxylase test:
a) Acidic condition: this is by fermenting dextrose that is in the medium.
Decarboxylase enzyme needs acidic medium to work. The low pH and the
presence of the amino acid will cause the organism to begin
decarboxylation. If the organism ferments dextrose, yellow color is seen. If
the organism produces a specific decarboxylase, the color turns from yellow
to purple color.
b) Anaerobic condition: this is adding mineral oil. This promotes
fermentation by locking out oxygen, and it also prevents false alkalinization
at the surface of the medium.
- Negative result: if the inoculated medium is yellow, or if there is no color
change, the organism is decarboxylase-negative for that amino acid.
- Positive result: if the medium turns purple, the organism is
decarboxylase-positive for that amino acid.
31
Decarboxylase Test Yellow Color or No Color Change (Negative) Purple Color (Positive) 32
Exercise:
1) Proteus and Salmonella are NLF and produce H2S (black center on
XLD). How can you differentiate between them?
2) How can you differentiate between Salmonella and Citrobacter on
XLD?
3) Write 3 virulence factors and 3 diseases caused by E. coli, Salmonella,
Shigella, Proteus, and Klebsiella.
33
4) Write the following about E. coli:
a) Gram stain:
b) Media & Colony:
c) Biochemical Reactions:
5) Write the following about Klebsiella:
a) Gram stain:
b) Media & Colony:
c) Biochemical Reactions:
34
6) Write the following about Salmonella:
a) Gram stain:
b) Media & Colony:
c) Biochemical Reactions:
7) Write the following about Shigella:
a) Gram stain:
b) Media & Colony:
c) Biochemical Reactions:
35
8) Write the following about Proteus:
a) Gram stain:
b) Media & Colony:
c) Biochemical Reactions:
36
API 20E
- API 20E is a biochemical kit used for identification of Enterobacteriaceae.
Boat
Cupule
- Procedure:
a) Inoculate the organism in distilled water (dH2O) and vortex the tube.
b) Place sterile water in the boat in order to prevent the drying.
c) By using sterile plastic pipette:
- Fill the tube of any test without underline i.e. ONPG with the inoculated
water.
- Fill the tube of any test with underline i.e. ADH with the inoculated water
+ fill the cupule with paraffin oil.
37
Tube
- Fill the tube and cupule of any test with semi-square i.e. CIT with the
inoculated water.
d) Close the boat and incubate it at 37°C for 24 hours.
e) In the next day:
- Add one drop of TDA reagent in TDA test.
- Add one drop of James reagent in indole test.
- Add VP1 (KOH) & VP2 (α-Naphthol) reagents in VP test.
f) Read the results:
- Each 3 tubes will be read together. If positive results: the first tube = 1, the
second tube = 2, and the third tube = 4. If negative, the tube counts 0 (See
the image bellow)
38
Oxidase
always
negative
g) Search the number i.e. 5144572 in the book, and see what is the organism.
39
Tube
Positive
Negative
ONPG
Yellow
Colorless
ADH
Red or Orange
Yellow
LDC
Red or Orange
Yellow
ODC
Red or Orange
Yellow
CIT
Blue-Green or Blue
Pale Green or Yellow
H2S
Black Deposit
No Black Deposit
URE
Red
Yellow or Orange
TDA
Reddish brown after
Adding TDA reagent
Yellow after Adding
TDA Reagent
IND
Red Ring after Adding
James Reagent
Yellow after Adding
James Reagent
VP
Red after Adding VP1
& VP2 Reagents
(10 min)
Diffusion of the
pigment
Colorless after Adding
VP1 & VP2 Reagents
(10 min)
No Diffusion
Yellow
Blue or
Blue-Green
GEL
GLU
MAN
INO
SOR
RHA
SAC
MEL
AMY
ARA
40
Pseudomonas aeruginosa
- Gram negative, rods.
- Strictly aerobic.
- Oxidase negative.
- NLF organism.
- Grow at 37°C and 42°C.
- Cetrimide agar:
It is a selective medium for Pseudomonas aeruginosa.
Colony: large oval flat colonies with bluish green exopigment, as well as
they have a characteristic fruity smell.
41
- On MacConkey:
Colony: large oval flat NLF colorless colonies.
- Biochemical Reactions:
Oxidase Test à negative
TSI à K/K g- H2SO/F Test à O+/F- (Oxidative organism).
42
Exercise:
1) Write the following:
a) Gram stain:
b) Media & Colony:
c) Biochemical Reactions:
2) Write 3 virulence factors and 3 diseases caused by Psuedomonas.
3) What are the differences between Enterobacteriaceae and
Psuedomonas? (2 differences)
43
Haemophilus spp.
- Species: H. influenza, H. parainfluenzae, H. ducreyi.
- Gram Negative, coccobacilli.
- Aerobic organisms, needs 5% CO2 in order to enhance the growth.
- Oxidase positive.
- Catalase positive.
- H. influenzae
- This species needs X (haemin) and V (NAD or NADP) to grow.
- Blood agar contains X factor only, whereas chocolate agar contains X & V
factors.
- V factor is inside the RBCs, and becomes outside after heating the blood.
- Chocolate agar:
Colony: small convex, smooth, pale grey or transparent colonies.
44
- Blood agar ‘contains X factor only’:
Satellitism: ‘colonies are largest, nearest to the line growth of
Staphylococcus aureus’.
S. aureus produces hemolysin that hemolyse RBCs, making the V factor to
be outside the RBCs, and giving the chance for H. influenzae to grow.
H. influenzae
S. aureus
- Biochemical Reaction:
Catalase positive, oxidase positive, API NH.
- Growth around XV disc only on nutrient agar.
45
Growth around XV disc H. influenzae
X
V
XV
Media
-
-
+
Chocolate
agar
H.
-
+
+
parainfluenzae
H. ducreyi
Chocolate
Agar
+
-
+
Blood Agar
& Chocolate
Agar
46
Exercise:
1) Write the following
a) Gram stain:
b) Media & Colony:
c) Biochemical Reactions:
2) Write 3 virulence factors and 3 diseases caused by H. influenzae.
3) What diseases can be caused by H. parainfluenzae & H. ducreyi?
47
Campylobacter spp.
- Gram negative, small, delicate, spirally curved seagull shaped rods.
- Microaerophilic organism (needs 10% CO2).
- C. jejuni is the most common species that cause the disease.
- C. jejuni and C. coli are thermophilic organisms that grow at 42°C.
- Oxidase positive.
- Medium:
Skirrow’s medium (blood agar + antibiotics)
It is a selective medium used for isolation of Campylobacter spp.
Colony: non-haemolytic spreading, droplet-like and moist colonies.
Incubation: this organism is microaerophilic and it needs 10% CO2 by
using jar + microaerophilic gas pack. Incubation is at 42°C for 48 hours.
48
- Biochemical Reactions:
Oxidase à positive
Hippurate Test à C. jejuni (positive, purple), C. coli (negative)
Hippurate Test
Some bacteria can hydrolyze hippurate to the amino acid glycine and
benzoate by means of the enzyme hippuricase. Glycine can be detected with
ninhydrin (2,2-Dihydroxyindane-1,3-dione), which reacts with free amino
groups (-NH2) and a purple product is formed.
Enzyme: hippuracase.
End Products: glycine and benzoate.
Procedure
1.
2.
3.
4.
5.
Add 0.1 ml sterile distilled water (dH2O) in test and control tubes.
Add 5 colonies in both tubes.
Add hippurate disc in both tubes.
Incubate the tubes at 37ºC for 2 hours.
Add 2 drops of ninhydrin in the test tube, and 2 drops of saline in the
control tube without mixing.
49
6. Incubate both tubes at 37ºC for 30 minutes.
7. Read the result
•
•
Positive test result: purple color.
Negative test result: colorless.
50
Exercise:
1) Write the following
a) Gram stain:
b) Media & Colony:
c) Biochemical Reactions:
2) Write 3 virulence factors and the disease caused by C. jejuni.
51
Vibrio spp.
- Gram negative curved rods, comma-shaped bacterium.
- Motile organism.
- Facultative anaerobes (O+/F+).
- Oxidase positive.
- Non-lactose fermenters.
- Species:
V. cholerae à causes cholera.
V. Eltor à causes cholera.
V. parahaemolyticus à causes food poisoning.
V. vulnificus à causes wound infection and septicaemia.
- There are 2 types of Vibrio
1. Non-halophilic Vibrio:
They don’t need salt (NaCl) in the media to grow i.e. they can grow on
CLED (NLF ‘colorless’). However, they can grow in presence of salt.
Ex: V. cholerae
52
2. Halophilic Vibrio:
They need salt (NaCl) in the media to grow i.e. they cannot grow on CLED.
Ex: V. parahaemolyticus
- Medium:
Alkaline Peptone Water
It is an enrichment medium for Vibrio spp.
Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS)
- It is a selective medium for isolating Vibrio spp.
- Indicator: thymol blue and bromothymol blue.
- Colony:
V. cholerae à large dome, yellow colonies (ferment sucrose).
53
V. parahaemolyticus à large dome, green colonies (doesn’t ferment
sucrose).
- Biochemical Reactions of V. cholerae:
Oxidase positive, catalase positive, indole positive, nitrate reduction test
positive, ferment sucrose, TSI (
).
54
Exercise:
1) Write the following about V. cholerae
a) Gram stain:
b) Media & Colony:
c) Biochemical Reactions:
2) Write 3 virulence factors and the disease caused by V. cholerae
3) What is the difference between Vibrio and Enterobacteriaceae?
4) What are the differences between Vibrio and Pseudomonas?
55
Clostridia
- Gram positive, bacilli.
- Anaerobes.
- Spore-formers
- Sensitive to metronidazole.
-Species:
C. perfringens à causes gas gangrene, food poisoning
C. tetani à causes tetanus, neonatal tetanus.
C. difficile à causes pseudomembranous colitis.
C. perfringens
- Gram positive bacilli, brick shape or carbox shape with subterminal spores.
- Media:
Blood agar ‘colony’: large, mucoid, grey colonies with double zone of
β-haemolysis after incubation anaerobically (using anaerobic gas pack) at
37°C for 48 hours.
56
- Litmus Milk “purple color”
C. perfringens gives stormy milk or stormy clot because it produces acid and
gas resulting from fermentation of lactose and clotting casein that present in
milk.
57
C. tetani
- Gram positive, bacilli, with terminal spores, drum stick appearance.
- Media
Blood agar ‘colony’: fine branching projections, the central part of colonies
becomes slightly raised (ground glass appearance) + offensive smell, after
incubation anaerobically (using anaerobic gas pack) at 37°C for 48 hours.
C. difficile
- Media
Cycloserine Cefoxitin Fructose Agar (CCFA)
Colony: round, flat, opaque colonies with typical horse manure smell, after
incubation anaerobically (using anaerobic gas pack) at 37°C for 48 hours.
58
- Colonies give green-yellow fluorescence under long wave ultra-violet light
(365 nm) in a dark room. Furthermore, C. difficile identity was confirmed by
using latex agglutination test according to the manufacturer’s instructions.
- Rapid tests e.g. C. difficile quick check complete®
*Before culturing stool sample for isolating C. difficile & C. perfringenes,
the stool sample will be heated at 80ºC for 15 min in order to kill other
organisms.
59
Exercise:
1) Write 3 virulence factors and diseases caused by C. perfringens, C.
tetani and C. difficile
60
Bacteroides spp.
- Gram negative rods (short rods).
- Anaerobe.
- Non-motile.
- Sensitive metronidazole.
-Non-spore former.
- Media
Neomycin Blood Agar ‘selective medium’ or blood agar
Colonies: grey, non-haemolytic, 1-3 mm in size glistening colonies after
incubation anaerobically (using anaerobic gas pack) at 37°C for 48 hours.
61
- Bacteroides fragilis à esculin broth positive
62
Exercise:
1) Write 3 virulence factors and diseases caused by Bacteroides fragilis
2) How can you differentiate between anaerobes and facultative
anaerobes?
63