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Transcript of Appendix
Appendix I
Tribhuvan University Teaching Hospital, Institute of medicine,
Department of microbiology
CLINICAL AND MICROBIOLOGICAL PROFILE OF PATEINT
Serial no: lab no: Date:
Clinical profile
Name: Age: sex: M F
Hospitalized: Yes No If no, duration of last hospital admission
If yes, duration of hospital admission
Living Status: Rural Urban
Habit: Smoker Non smoker others:
Clinical Features: (as per physician)
a) pulmonary infiltration on chest radiograph : Yes No
b) fever: Yes No c) cough: Yes No
d) purulent sputum production: Yes No e) Leucocytosis: Yes No
Patient on antibiotics: yes if yes, Antibiotics taken: Duration:
No others if any
Microbiological profile
Specimen: Sputum collection time: Time of lab receipt:
Day 1
Macroscopic observation:
a)Purulent b) Muco-purulent c)Mucoid d)Mucosalivary e)Watery f)others
Microscopic observation (Gram's stain):
Day 2
Colony characteristics:
Blood Agar: Chocolate Agar:
Mac Conkey Agar:
Gram’s stain results:
Catalase: Oxidase: Coagulase: Others:
Provisional Identification:
Biochemical test performed.
Day 3
Interpretation of biochemical tests:
a) TSI: b) SIM: c)Citrate: d)Urease: e) X-V factor dependence test: f)Serotyping if needed: g)Others:
Organism identified as:
Antibiotic sensitivity profile of the isolate :( disk diffusion Method)
Antibiotics ZOI of control (mm) ZOI of test (mm) Interpretation
Comments on Drug Resistance Pattern: MDR Non MDR
Comments on MRSA (Cefoxitin test):
Comments on ESBL production:
Comments on MBL production:
Performed by: Checked by:
Appendix-III
List of equipments and materials used during the study:
A. List of Equipments:
1. Hot air oven: Sakura (Japan)
2. Incubator: Sanyo (Japan)
3. Autoclave: Sakura (Japan)
4. Refrigerator: Toshiba (Japan)
5. Centrifuge: Hitachi (Japan)
6. Weighing Machine: Chyo MP 300 (Japan)
7. Water distillation Plant: Yamato (Japan)
8. Laminar Flow: Dalton (USA)
9. Water bath: NSW (India)
10. Carbondioxide incubator: Yamato (Japan)
11. Electronic balance: Chyo MP (Japan)
12. Vortex mixer: Yamato (Japan)
B. List of Materials
1. Sterile gloves
2. Sterile test tubes with sterile swabs
3. Labelling stickers
4. Inoculating loops
5. Straight wire
6. Glass slides
7. Beakers
8. Test tubes
9. Flasks
10. Pipettes
11. Measuring cylinders
12. Forceps
13. Dropping bottles
14. Wash bottle
15. Glass rods
Antibiotic discs
All the antibiotics discs used for the susceptibility tests were from Oxoid Unipath Ltd.
Basingstoke, Hampsire, England. The antibiotics used were as follows:
1. Amoxycillin /Ampicillin (10µg)
2. Amoxycillin plus Clavulanic acid (30µg/10ug)
3. Amikacin (30ug) (B.D. USA)
4. Azithromycin(15ug) (B.D. USA)
5. Carbencillin(100ug) (B.D. USA)
6. Ciprofloxacin (5µg) (B.D. USA)
7. Ofloxacin (5µg) (B.D. USA)
8. Chloramphenicol ((30µg) (B.D. USA)
9. Cotrimoxazole (1.25/23.75µg)
10. Ceftriaxone (30µg) (B.D. USA)
11. Cefotaxime (30µg) (B.D. USA)
12. Ceftazidime(30µg) (B.D. USA)
13. Cefoperazone-sulbactum
14. Cefoxitin(30ug) (B.D. USA)
15. Cefipime (30µg) (B.D. USA)
16. Clindamycin(2ug) (B.D. USA)
17. Cloxacillin
18. Colistin Sulphate(10ug) (B.D. USA)
19. Doxycycline(30ug) (B.D. USA)
20. Erythromycin(15ug) (B.D. USA)
21. Imipenem(10µg) (B.D. USA)
22. Meropenem(10ug) (B.D. USA)
23. Oxacillin(1ug) (B.D. USA)
24. Piperacillin(100ug) (B.D. USA)
25. Piperacillin –Tazobactum(100/10ug) (B.D. USA)
26. Polymyxin B (300U) (B.D. USA)
27. Tobramycin(10ug) (B.D. USA)
28. Vancomycin(30ug) (B.D. USA)
Lists of Chemicals
i. Crystal violet Qualigens, India
ii. Iodine crystals Qualigens, india
iii. Potassium iodide Qualigens, India
iv. Acetone Qualigens, India
v. Ethanol Qualigens, India
vi. Safranine Qualigens, India
vii. Immersion oil Qualigens,India
viii. Methanol Qualigens, India
ix. Sodium chloride powder Qualigens, India
x. Sodium dithiothreitol Qualigens, India
xi. EDTA Sigma chemicals, St. Louis, MO
The chemicals were all of analytical grade and purchased from local suppliers or made available
from TUTH.
Appendix-IV
I. Composition and preparation of different culture media
The culture media used were from two companies
A. Hi-Media Laboratories Pvt. Limited, Bombay, India
B. Oxoid Unipath Ltd. Basingstoke, Hampsire, England
(All compositions are given in grams per liter and at 25ºC temperature)
1. Blood agar base (Oxoid, England)
Blood agar base (Infusion agar) + (5 -10%) sheep blood
Ingredients gm/liter
Protease peptone 15.0
Liver Digest 2.5
Yeast extract 5.0
Sodium Chloride 5.0
Agar 12.0
Final pH (at 25ºC) 7.3 ± 0.2
Preparation: 40 grams of the blood agar base medium was suspended in 1000 ml distilled water,
dissolved by boiling and sterilized by autoclaving at 121ºC (15 lbs pressure) for 15 minutes.
After cooling to 45-50ºC, 5-7% sterile defibrinated sheep blood was added aseptically, then
mixed with gentle rotation and immediately poured in sterile petri plates.
2. Chocolate agar
Preparation: It was prepared as described for blood agar except after adding the blood; the media
was heated in a 70˚C water bath until it became brown in color. After allowing the medium to
cool to about 45˚C, it was mixed and dispensed in sterile petri dishes as described for blood agar.
3. MacConkey Agar (Oxoid, England)
Ingredients gm/liter
Peptone 20.0
Lactose 10.0
Bile salts 5.0
Sodium chloride 5.0
Neutral Red 0.075
Agar 12.0
Final pH (at 25ºC) 7.4±0.2
Preparation: 52 grams of the medium was suspended in 1000 ml of distilled water and then
boiled to dissolve completely. Then the medium was sterilized by autoclaving at 121ºC (15 lbs
pressure) for 15 minutes. After cooling to 50ºC immediately poured in sterile petriplates.
4. Mueller Hinton Agar (Oxoid, England)
Ingredients gm/liter
Beef, infusion form 300.0
Casein Acid Hydrolysate 17.5
Starch 1.5
Agar 17.0
Final pH (at 25ºC) 7.4±0.2
Preparation: 38 grams of the medium was suspended in 1000 ml distilled water and the medium
was warmed to dissolve completely. Then the medium was sterilized by autoclaving at 121ºC (15
lbs pressure) for 15 minutes. After cooling to 50ºC immediately poured in sterile petriplates.
5. Nutrient Agar (Oxoid, England)
Ingredients gm/liter
Peptone 10.0
Sodium Chloride 5.0
Beef Extract 10.0
Yeast Extract 1.5
Agar 12.0
Final pH (at 25ºC) 7.4±0.2
Preparation: 37 grams of the medium was suspended in 1000 ml of distilled water and then
boiled to dissolve completely. Then the medium was sterilized by autoclaving at 121ºC (15 lbs
pressure) for 15 minutes
6. Nutrient Broth (Hi-Media)
Ingredients gm/liter
Peptone 5.0
Sodium Chloride 5.0
Beef Extract 1.5
Yeast Extract 1.5
Final pH (at 25ºC) 7.4±0.2
Preparation: 13 grams of the medium was dissolved in 1000 ml distilled water and autoclaved at
121ºC for 15 minutes. After cooling, poured in sterile tubes and bottles.
7. Peptone Water (Oxoid, England)
Ingredients gm/liter
Peptone 10
Sodium Chloride 5
Preparation: 15 grams of the medium was dissolved in 1000 ml distilled water and autoclaved at
121ºC for 15 minutes. After cooling, poured in sterile tubes and bottles.
II. Composition and preparation of different biochemical tests media
1. MR-VP Medium (Hi-Media laboratories)
Ingredients gm/liter
Buffered Peptone 7.0
Dextrose 5.0
Dipotassium Phosphate 5.0
Final pH (at 25ºC) 6.9±0.2
Preparation: 17 grams was dissolved in 1000 ml distilled water. 3 ml of medium was distributed
in each tube and autoclaved at 115ºC for 15 minutes.
2. Hugh and Leifson's Medium (Hi- media laboratories)
Ingredients gm/liter
Tryptone 2.0
Sodium chloride 5.0
Dipotassium Phosphate 0.3
Bromothymol Blue 0.08
Agar 2.0
Final pH (at 25ºC) 6.8±0.2
Preparation: 9.4 grams of the medium was rehydrated in 1000 ml cold distilled water and then
heated to boiling to dissolve completely. The medium was distributed in 100 ml amounts and
sterilized in the autoclave for 15 minutes at 15 lbs pressure (at 115ºC). To 100 ml sterile medium
aseptically added 10 ml of sterile Dextrose and mixed thoroughly and dispensed in 5 ml
quantities into sterile culture tubes.
3. Sulphide Indole Motility (SIM) medium (Oxoid, England)
Ingredients gm/liter
Tryptone 20.0
Peptone 6.1
Ferrous ammonium sulphate 0.2
Sodium Thiosulphate 0.2
Agar 3.5
Final pH (at 25ºC) 7.3±0.2
Preparation: 30 grams of the medium was suspended in 1000 ml distilled water and dissolved
completely. Then it was distributed in tubes to a depth of about 3 inches and sterilized by
autoclaving at 115ºC for 15 minutes.
4. Simmon's Citrate Agar (Oxoid, England)
Ingredients gm/liter
Magnesium Sulphate 0.2
Ammonium dihydrogen Phosphate 0.2
Sodium ammonium phosphate 1.0
Sodium Citrate, tribasic 2.0
Sodium Chloride 5.0
Agar 15.0
Bromothymol Blue 0.08
Final pH (at 25ºC) 6.8±0.2
Preparation: 23 grams of the medium was dissolved in 1000 ml distilled water. 3ml medium was
distributed in test tubes and sterilized by autoclaving at 115ºC for 15 minutes. After autoclaving
tubes containing medium were tilted to form slant.
5. Triple Sugar Iron Agar (TSI) (Oxoid, England)
Ingredients gm/liter
Lab-lemco powder 3.0
Yeast Extract 3.0
Peptone 20.0
Lactose 10.0
Sucrose 10.0
Glucose 1.0
Ferric Citrate 0.3
Sodium Chloride 5.0
Sodium Thiosulphate 0.3
Phenol Red 0.024
Agar 12.0
Final pH (at 25ºC) 7.4±0.2
Preparation: 65 grams of the medium was dissolved in 1000 ml of distilled water and sterilized
by autoclaving at 115ºC pressure for 15 minutes. The medium was allowed to set in slope form
with a butt about 2.5 inches of length.
6. Christensen Urea Agar (Oxoid, England)
Ingredients gm/liter
Peptone 1.0
Dextrose 1.0
Sodium Chloride 5.0
Dipotassium Phosphate 1.2
Mono-potassium phosphate 0.8
Phenol Red 0.012
Agar 15.0
Final pH (at 25ºC) 7.4±0.2
Preparation: 24 grams of the medium was suspended in 950 ml distilled water and sterilized by
autoclaving at 115ºC for 15 minutes. After cooling at about 45ºC, 50 ml of 40% urea was added
and mixed well. Then 5 ml was dispensed in test tube and set at slant position.
7. Phenylalanine deaminase medium (Oxoid, England)
Ingredients gm/liter
Yeast extract 3
L-phenylalanine 1
Disodium hydrogen phosphate 1
Sodium chloride 5
Agar 12
Final pH (at 25ºC) 7.4±0.2
Preparation: 22 grams of the medium was dissolved in 1000 ml distilled water. 3ml medium was
distributed in test tubes and sterilized by autoclaving at 115ºC for 15 minutes. After autoclaving
tubes containing medium were tilted to form slant.
8. Amino acid decarboxylase test medium (Oxoid, England)
Ingredients gm/liter
Peptone 5
Meat extracts 5
Glucose 0.5
Pyridoxal 0.005
Bromocresol purple (1 in 500 solution) 5ml
Cresol red (1 in 500 solution) 2.5 ml
Preparation: Dissolved the solids in water and adjusted the pH to 6.0±0.2 before the addition of
the indicators. This was the basal medium and to it was added the amino acid whose
decarboxylation was to be tested. Divided the basal medium into four portions and treated
separately as follows:
1. added 1% L- lysine hydrochloride
2. added 1% L- ornithine hydrochloride
3. added 1% L- arginine hydrochloride
4. No additions (Control)
The pH was readjusted the pH to 6.0±0.2. Distributed 1 ml quantities in small tubes containing
sterile liquid paraffin to provide a layer about 5 mm thick above the medium. Autoclaved at
115ºC for 15 minutes.
III. Composition and preparation of different staining and tests reagent
1. Preparation of Gram stain Reagent
(a) Crystal violet solution (Hucker's crystal violet)
I. Crystal violet stock solution
Crystal violet (90% to 95% dye content) 40 g
Ethanol, 95% 400 ml
Preparation: Dissolved and mixed in a glass bottle, labeled with a 1-year expiration date, and
stored at room temperature.
II. Ammonium oxalate solution (1%)
Ammonium oxalate (reagent grade) 16 g
Distilled water 1600 ml
Preparation: Dissolved and mixed in a brown glass bottle, labeled with a 1-year
expiration date, and stored at room temperature.
III. Crystal violet working solution
Crystal violet stock solution 40 ml
Ammonium oxalate solution (1%) 160 ml
Preparation: Filtered crystal violet stock solution into a glass. Allowed to filter
completely, and then filtered ammonium oxalate solution. Labeled with earliest
expiration date of stock solutions.
(b) Gram's iodine
I. Stock lugol's iodine solution
Iodine crystals (reagent grade) 25 g
Potassium iodide (reagent grade) 50 g
Distilled water 500 ml
Preparation: Mixed and let stand until dissolved in a brown glass bottle, labeled with a 6- month
expiration date, and stored at room temperature.
II. Sodium bicarbonate, 5% (w/v)
Sodium bicarbonate (reagent grade) 50 g
Distilled water 1000 ml
Preparation: Dissolved in a glass bottle, labeled with a 1-year expiration date, and stored at room
temperature.
III. Gram's iodine
Stock Lugol's iodine solution 60 ml
Ditilled water 220 ml
Sodium bicarbonate (5%) 60 ml
Preparation: Mixed in a brown glass bottle, labeled with a 6-month expiration date, and stored at
room temperature.
(c) Acetone-Alcohol Decolorizer
Acetone 500 ml
Ethanol (absolute) 475ml
Distilled Water 25 ml
Preparation: To 25 ml D/W, 475 ml of absolute alcohol was added, mixed and transferred into a
clean bottle. Then immediately, 500 ml acetone was added to the bottle and mixed well.
(d) Safranin (Counter Stain)
Safranin 10.0 gm
Distilled Water 1000 ml
Preparation: In a clean piece of paper, 10 gm of safranin was weighed and transferred to a clean
bottle. Then 1 liter D/W was added to the bottle and mixed well until safranin dissolved
completely.
2. Normal Saline
Sodium Chloride 0.85 gm
Distilled Water 100 ml
Preparation: The sodium chloride was weighed and transferred to a leak- proof bottle premarked
to hold 100 ml. Distilled water was added to the 100 ml mark, and mixed until the salt was fully
dissolved. The bottle was labeled and stored at room temperature.
Biochemical Tests Reagents
a. For Catalase Test
Catalase Reagent (3% H2O2)
Stock Hydrogen Peroxide (3%) 3ml
Distilled Water 97ml
Preparation: To 90 ml of D/W, 10 ml of hydrogen peroxide was added and mixed well
b. For Oxidase Test
Oxidase Reagent (impregnated in Whatman's No. 1 filter paper)
Tetramethyl p-phenylenediamine dihydrochloride (TPD) 1 gm
Distilled Water 100 ml
Preparation: This reagent solution was made by dissolving 1 gm of TPD in 100 ml D/W. To that
solution strips of Whatman's No.1 filter paper were soaked and drained for about 30 seconds.
Then these strips were dried in incubator and stored in a dark bottle tightly sealed with a screw
cap.
c. For indole Test
Kovac's Indole Reagent
Isoamyl alcohol 30 ml
p-dimethyl aminobenzaldehyde 2.0 gm
Hydrochloric acid 10 ml
Preparation: In 30 ml of isoamyl alcohol, 2 gm of p-dimethyl aminobezaldehyde was dissolved
and transferred to a clean brown bottle. Then to that, 10 ml of conc.HCL was added and mixed
well.
d. For Methyl Red Tests
Methyl Red Solution
Methyl red 0.05 gm
Ethyl alcohol (absolute) 28 ml
Distilled water 22 ml
Preparation: To 28 ml ethanol, 0.05 gm of methyl red was dissolved and transferred to a clean
brown bottle. Then 22 ml of D/W was added to that bottle and mixed well.
e. For Voges- Proskauer Test (Barritt's Reagent)
Solution A
Alpha-Naphthol 5.0 gm
Ethyl alcohol (absolute) 100 ml
Preparation: To 25 ml D/W, 5 gm of alpha-Naphthol was dissolved and transferred into a clean
brown bottle. Then the final volume was made 100 ml by adding D/W.
Solution B
Potassium hydroxide (KOH) 40.0 g
Distilled water 1000 ml
Preparation: To 25 ml D/W, 40 gm of KOH was dissolved and transferred into a clean brown
bottle. Then the final volume was made 100 ml by adding D/W.
4. McFarland tube (No. 0.5)
0.5 ml of 0.048 M Bacl2 (1.17% w/v Bacl2H2O) was added to 99.5 ml of 0.18 M H2SO4 (1%
w/v) with constant stirring. The McFarland standard was thoroughly mixed to ensure that it is
evenly suspended. Using matched cuvettes with a 1 cm light path and water as a blank standard,
the absorbance was measured in a spectrophotometer at a wavelength of 625 nm. The acceptable
range for the turbidity standard is 0.08-0.13. The standard was distributed into screw-cap tubes
of the same size and volume as those used to prepare the test inoculum. The tubes were sealed
tightly to prevent loss by evaporation and stored protected from light at room temperature. The
turbidity standard was then vigorously agitated on a vortex mixer before use. Standards may be
stored for up to 6 months, after which they should be discarded.
Appendix-V
Gram staining procedure
The test was originally developed by Christian Gram in 1884.The modification currently used for
general bacteriology was developed by Hucker in 1921. Gram-stain can be used effectively to
divide all bacterial species into two large groups: those that take up the basic dye, crystal violet
(Gram-positive) and those that allow the crystal dye to wash out easily with the decolourizer
alcohol or acetone (Gram-negative). The following steps are involved in Gram stain:
1. A thin film of the material to be examined was prepared and dried.
2. The material on the slide was heat fixed and allowed to cool before staining.
3. The slide was flooded with crystal violet stain and allowed to remain without drying for
30 to 60 seconds.
4. The slide was rinsed with tap water, shaking off excess.
5. The slide was flooded with iodine solution and allowed to remain on the surface with out
drying for twice as long as the crystal violet is in contact with the slide surface.
6. The slide was rinsed with tap water, shaking off excess.
7. The slide was flooded with acetone alcohol decolorizer for 10 seconds and rinsed
immediately with tap water until no further colours flow from the slide with the
decolourizer. Thicker smear requires more aggressive decolorizing.
8. The slide was flooded with counter stain (safranine) for 30 seconds and washed off with tap
water.
9. The slide was blotted between two clean sheets of blotting paper and examined
microscopically under oil immersion at 100X.
Appendix-VI
Methodology of biochemical tests used for identification of bacteria
A. Catalase test
Bacteria that synthesize the enzyme catalase hydrolyze hydrogen peroxide into water and
gaseous oxygen, which results in the liberation of gas bubbles. During aerobic respiration, in the
presence of oxygen, microorganism produce hydrogen peroxide, which is lethal to the cell itself.
The enzyme catalase is present in most aerobic and facultative anaerobic bacteria containing
cytochrome, the main exception being Streptococcus spp. Reduced flavoprotein reacts directly
with gaseous oxygen by way of electron reduction to form hydrogen peroxide, which is an
oxidative end product of the aerobic breakdown of sugars.
FPH2 + O2 FP + H2O2
(Reduced Flavoprotein) (Oxidized Flavoprotein)
Hydrogen peroxide thus formed is toxic to bacteria. Catalase enzyme breaks down hydrogen
peroxide into water and oxygen
Catalase
2H2O2 2H2O +O2
Procedure: Touched the center of an 18 to 24 hour, well isolated colony to a glass slide and one
drop of 3% H2O2 was put on the surface of the slide. The positive test was indicated by the
formation of active bubbling of the oxygen gas immediately. A false positive reaction may be
obtained if the culture medium contains catalase (e.g. Blood Agar) or if an iron wire loop is used.
B. Oxidase test
In the presence of atmospheric oxygen, a bacterium's intracellar cytochrome oxidase enzymes
oxidize the phenylenediamine reagent (an electron acceptor) to form a deeppurple compound,
indole phenol. This oxidase reaction is due to the presence of a cytochrome oxidase system
which activates the oxidation of reduced cytochrome by molecular oxygen, which in turn acts as
an electron acceptor in the terminal stage of the electron transfer system. Some of organisms
capable of growing in the presence of oxygen i.e. aerobic or facultative anaerobic organisms are
oxidase positive and at the same time they produce the enzyme catalase. Obligate anaerobic
organisms lack oxidase activity since they are unable to live in the presence of atimospheric
oxygen and do not possess a cytochrome oxidase system. The test is used for screening species
of Neisseria, Alcaligenes, Aeromonas, Vibrio, Campylobacter and Pseudomonas which give
positive reactions and for the excluding the Enterobacteriaceae, all species of which give
negative reactions.
Procedure: A piece of filter paper was soaked with few drops of oxidase reagent (watman's No. 1
filter paper impregnated with 1% tetramethyl-p-phenylene diaminedihydrochloride). Then the
colony of the test organism was smeared on the filter paper. The positive test was indicated by
the appearance of blue-purple color within 10 seconds.
C. Oxidation-Fermentation test
This method (Hugh & Leifson 1953) depends upon the use of a semi-solid tubed medium
containing the carbohydrate (usually glucose) together with a pH indicator. If acid is produced
only at the surface of the medium, where conditions are aerobic, the attack on the sugar is
oxidative. If acid is found throughout the tube, including the lower layers where conditions are
anaerobic, the breakdown is fermentative.
Procedure: The test organism was stabbed into the bottom of two sets of tubes with Hugh and
Leifson's media, bromothymol blue being the pH indicator. The inoculated medium in one of the
tubes was covered with a 10 mm deep layer of sterile paraffin oil. The tubes were then incubated
at 37ºC for 24 hours. After incubation the tubes were examined for carbohydrate utilization as
shown by yellow color of media that denotes the acid production. Fermentative organism utilizes
the carbohydrate in both the open and sealed tubes as shown by a change in color of the medium
from green to yellow. Oxidative organisms, however, are able to use the carbohydrate only in the
open tube.
D. Indole Production test
The ability of an organism to split indole from the amino acid tryptophan is due to the presence
of tryptophanase. Indole, if present, combines with the aldehyde in the reagent to produce a pink
to red-violet quinodal compound (benzaldehyde reagent). The enzyme tryptophanase catalyses
the deamination reaction attacking the tryptophan molecule in its side chain and leaving the
aromatic ring intact in the form of indole.
Procedure: A smooth bacterial colony was stabbed on SIM (Sulphide Indole Motility) medium
by a sterile stab wire and the inoculated media was incubated at 37ºC for 24 hours. After 24
hours incubation, 2-3 drops of Kovac's reagent (paradimethylaminobenzaldehyde in acid
ethanol) was added. Appearance of red color on the top of media indicates indole positive. Indole
if present combines with the aldehyde present in the reagent to give a red color in the alcohol
layer. The color reaction is based on the presence of the pyrrole structure present in indole.
E. Methyl Red test
This test is used to determine if an organism is able to produce and maintain stable acid end
product from glucose fermentation Methyl red indicator (red color below pH 4.4; yellow color at
pH 5.8) is used to determine the pH after an enteric gram negative rod has fermented glucose to
completion. All members of the Enterobactericeae give a positive methyl red reaction when
tested up to 24 hour due to conversion of glucose to pyruvic acid by the Embden-Meyerhof
pathway. After further incubation (2 to 5 days) those organisms that are MR positive continue to
metabolize pyruvic acid to lactic, acetic, and formic acids by the mixed acid pathway and are
able to maintain the acid pH (<4.4). Medium used was MR/VP broth, pH 6.9. Methyl red is an
indicator which is already acid and will denote change in degree of acidity by color reactions
over a pH range of4.4-6.0.
Procedure: A pure colony of the test organism was inoculated into 2 ml of MR/VP medium and
was incubated at 37ºC for 24 hours. After incubation, about 5 drops of methyl red reagent was
added and mixed well. The positive test was indicated by the development of bright red color,
indicating acidity and negative with yellow color.
F. Voges-Proskauer (VP) test
The voges-Proskauer (VP) test is used to determine if an organism produces
acetylmethylcarbinol, a neutral end product (acetoin) or its reduction product 2, 3 butanidiol
during fermentation of carbohydrates. If present, acetylmethylcarbinol is converted to diacetyl in
the presence of a-naphthol, strong alkali (40% KOH), and atmospheric oxygen.The á-naphthol is
not the part of the original procedure but is found to act as a color intensifier by Barrit and must
be added first. The diacetyl and quanidine-containing compounds found in the peptones of the
broth then condense to form a pinkish red polymer. An organism of the enterobacterial group is
usually either methyl red positive, Voges-proskauer negative or methyl red negative, Voges-
proskauer positive. The voges proskauer test for acetoin is used primarily to separate E.coli from
Klebsiella and Enterobacter species.
Procedure: A pure colony of the test organism was inoculated into 2 ml of MR/VP medium and
was incubated at 37ºC for 24 hours. After incubation, about 5 drops of Barritt's reagent was
added and shaken well for maximum aeration and kept for 15 minutes, positive test is indicated
by the development of pink red color.
G. Citrate Utilization test (Simmons)
Citrate agar is used to test an organism's ability to utilize citrate as a source of energy. The
medium contains citrate as the sole carbon source and inorganic ammonium salts as the sole
source of nitrogen. Growth is indicative of utilization of citrate, an intermediate metabolite in the
Kreb's cycle. In bacteria the cleavage of citrate involves an enzyme system without the
intervention of the coenzyme A; this enzyme is called citratase or citrate desmolase.When the
bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which increases
alkalinity. The shift in pH turns the bromothymol blue indicator in the medium from green to
blue above pH 7.6. This medium is recommended as part of differentiating among the species of
Enterobacteriaceae.
Procedure: Streaked the slant back and forth with a light inoculums picked from the center of a
well –isolated colony and incubated at 37ºC for 24 hours. A positive test was indicated by the
growth of organism and change of media by green to blue, due to alkaline reaction. The pH
indicator bromothymol blue has a pH range of 6.0-7.6, i.e. above pH 7.6; a blue color develops
due to alkalinity of the medium.
H. Motility test
The motility test is used to detect the presence of flagella by bacteria, allowing them to travel in
and out of the microscopic field or beyond their initial inoculation in agar. This test is done to
determine if an organism is motile or non-motile. Bacteria are motile by means of flagella occur
primarily among the bacilli; however a few cocci forms are motile. Motile bacteria may contain
single flagella. The motility media used for motility tests are semisolid, making motility
interpretations macroscopic.
Procedure: Motility of organism was tested by hanging drop preparation and cultural method. In
cultural method, the test organism was stabbed in the SIM medium and incubated at 37ºC for 48
hours. Motile organisms migrate from the stab line and diffuse into the medium causing
turbidity. Whereas non motile bacteria show the growth along the stab line and the surrounding
media remains colorless and clear.
I. Triple sugar Iron (TSI) Agar Test
The TSI agar contains casein and meat peptones, phenol red as the pH indicator, 0.1% glucose,
1% lactose and 1% sucrose for fermentation. Ferric or ferrous ions and sodium thiosulphate are
present to detect hydrogen sulphide production. Organisms that are non-lactose fermenting
initially produce a yellow slant due to the production of acid from the glucose. The small amount
of glucose is rapidly depleted. Oxidative metabolism continues in the slant after the low
concentration of glucose has been depleted, producing an alkaline pH from the aerobic
breakdown of peptone; the slant turns red. There is no oxygen penetration into the butt and no
oxidative metabolism; the butt remains acid and yellow. Thus, a non-lactose fermenting
organism yields an alkaline (K) slant over an acid (A) butt (K/A); red slant; yellow butt). Lactose
fermenting and/or sucrose-fermenting bacteria continue to produce a large amount of acid in the
slant and butt so the reaction in both remains acid (A/A; yellow slant; yellow butt). If the slant
and butt remain neutral, the organism is not capable of fermenting glucose or other sugars (K/K;
red slant; red butt). Gas production from sugar fermentation is indicated by bubbles, fracturing of
the medium, or displacement of the medium.
Hydrogen sulphide is produced by the action of the bacteria with sodium thiosulphate. This is
detected by the reduction of ferric ions to produce a black precipitate.
Procedure: The test organism was stabbed and streaked on the surface of TSI and incubated at
37ºC for 24 hours. Acid production limited only to the butt region of the tube is indicative
glucose utilization, while acid production in slant and butt indicates sucrose or lactose
fermentation. The results were interpreted as follows:
a. Yellow (Acid)/ Yellow (Acid), Gas, H2S---Lactose/Sucrose fermenter, H2S producer.
b. Red (Alkaline)/ Yellow (Acid), No gas, No H2S - Only Glucose, not Lactose/Sucrose
fermenter, anaerogenic, No H2S production.
c. Red (Alkaline)/ No change - Glucose, Lactose and Sucrose Non fermenter.
d. Yellow (Acid)/ No change--- Glucose oxidizer
e. No change/No change--- Non fermenter.
J. Urea Hydrolysis test: (Christensen's urea agar)
Urea medium, whether a broth or agar, contains urea and pH indicator phenol red. Many
organisms, especially those that infect the urinary tract, have a urease enzyme, which is able to
split urea in the presence of water to release two molecules of ammonia and carbon dioxide. The
ammonia combines with the carbon dioxide and water to form ammonium carbonate, which
turns the medium alkaline, turning the indicator from its original orange-yellow color to bright
pink.
Procedure: The test organism was inoculated in a medium containing urea and the indicator
phenol red. The inoculated medium was incubated at 37ºC overnight. Positive organism showed
pink red color due to the breakdown of urea to ammonia. With the release of ammonia the
medium becomes alkaline as shown by a change in color of the indicator to pink.
K. Phenylalanine Deaminase Test
The phenylalanine deaminase (PDA) test is used to differentiate among the urease positive gram-
negative bacilli (Proteus, Providencia and Morganella) based on the ability of the
microorganisms to produce phenylpyruvic acid by oxidative deamination. Phenylalanine is an
amino acid that, upon deamination by oxidase enzymes, results in the formation of
phenylpyruvic acid. The deamination of phenylalanine to phenylpyruvic acid is detected by the
addition of a ferric chloride solution that acts as chelating agent with the á-keto acid by-product
to produce a light to deep green cyclic compound.
Procedure: Prior to inoculation, allowed the medium to equilibriate to room-
temperature. Using a heavy inoculum from an 18 to 24 hour pure culture, streaked the slant
surface using a fishtail motion. Incubated the inoculated slant aerobically at 35ºC for 18 to 24
hour. Following incubation, applied 4 to 5 drops of ferric chloride directly to the slant. Gently
rolled the reagent over the slant to dislodge surface colonies observed for the development of a
green color within 1 to 5 minutes.
L. Decarboxylase – Dihydrolase Tests
Arginine, lysine, and ornithine decarboxylase media are used to detect an organism's ability to
decarboxylate or hydrolyze an amino acid, forming an amine that produces an alkaline pH. The
basal medium is usually Moller's formula and contains meat peptones and beef extract, which
supply nitrogenous nutrients to support bacterial growth. Glucose is the fermentable
carbohydrate. The pH indicators are bromocresol purple and cresol red. Pyridoxal is an enzyme
cofactor which enhances decarboxylase activity. Arginine, lysine and ornithine are amino acids
that are singly added to basal medium to detect the production of enzymes which decarboxylate
or hydrolyze these substrates. If an organism in the medium ferments glucose, acids are produced
that lower the pH, resulting in a color change from purple to yellow. If decarboxylation or
hydrolysis of the amino acid occurs in response to the acid pH, alkaline end products (amines)
are formed which result in the medium reverting to its original color (purple). When the
organism does not ferment glucose, the medium does not turn yellow, the test can still be
performed, but it is important to include a control without amino acids for comparison.
Decarboxylation of lysine yields cadaverine, decarboxylation of ornithine yields putrescine, and
decarboxylation of arginine yields agmatine, which is hydrolyzed by a dihydrolase to form
putrescine. In another reaction arginine dihydrolase converts arginine to citruline, which is
converted to ornithine and then to putrescine. Since decarboxylation is an anaerobic reaction, the
contents of each tube must be sealed with oil or paraffin.
Procedure: Inoculated each broth being tested with one or two colonies from an 18 to 24 hour
culture. For non –glucose-fermenting, gram-negative rods, control tube included. Overlayed the
inoculated tubes with approximately 1 ml of mineral oil to cover broth layer entirely without
introducing air. Tightened the cap on the tubes. Incubated aerobically at 35ºC for at least 18 hour
and up to 7days, and observed daily for purple color.
M. Coagulase test
S. aureus, the most pathogenic of the staphylococci, is separated from other species by the
presence of coagulase. Coagulase is a thermostable thrombin like substance that activates
fibrinogen to form fibrin, resulting in a fibrin clot. This is demonstrated in the test tube by the
formation of a clot when plasma is inoculated with the staphylococcus. The substance is known
as free coagulase, since it is liberated by the cell. In most , but not all , S. aureus organisms,
fibrinogen binding cell surface receptor is also present in cell wall, called " bound coagulase" or
"clumping factor". Clumping factor is demonstrated by the ability of the organism to act directly
on the fibrinogen in the plasma to clump it in a slide assay. This test is used specifically to
differentiate species within the genus. S. aureus (Usually Positive) from S. saprophyticus, S.
epidermidis (negative). A positive coagulase test is usually the final diagnostic criterion for the
identification of S.aureus.
a. Slide Coagulase Test:
Procedure: For slide coagulase test, a drop of physiological saline was placed on three places of a
slide, and then a colony of the test organism was emulsified in two of the drops to make thick
suspensions. Later a drop of plasma was added to one of the drops to make thick suspensions.
Then a clumping was observed within 10 second for the positive coagulase test. No plasma was
added in second suspension. This was used for the differentiation of any granular appearance of
the organism from true coagulase clumping. The third drop of saline was used for a known strain
of coagulase positive staphylococci
b. Tube Coagulase Test
This test is carried out to detect production of free coagulase. Plasma contains coagulase reacting
factor (CRF) which activates free coagulase. The activated coagulase acts upon prothrombin thus
converting it to thrombin. Thrombin converts fibrinogen into fibrin which is detected as a firm
gel (clot) in the tube test. Tube test is performed when negative or doubtful results are obtained
in slide coagulase test.
Procedure: In the tube coagulase test, plasma was diluted 1 in 10 in physiological saline Four
small tubes were taken, one for test organism, one for positive control, one for negative control,
and one to observe self clotting of plasma. Then 0.5 ml of the diluted plasma was pipetted into
each tube and 0.5 ml of test organism, 0.5 ml of positive control (S. aureus culture), and 0.5 ml
negative control (S. epidermidis culture) was added to three tubes, to the fourth tube, 0.5 ml
sterile broth was added. After mixing gently, all tubes were incubated at 37º on a water bath for 6
hours and observed for gel formation in every 30 minutes. The clotting is observed by gently
tilting the tube for positive coagulase test. If no clot was observed for 4 hours, then the tubes
were kept at room temperature overnight and next day, they were again observed for the clotting
of plasma.
N.Butyrate Esterase test
The butyrate test is a rapid test for the detection of the enzyme butyrate esterase. When used in
conjunction with characteristic morphology on blood agar, typical Gram stain and a positive
oxidase test, the butyrate test is useful for the definitive identification of M. catarrhalis.
Hydrolysis of bromochloro-indolyl butyrate (IB) substrate by the butyrate esterase releases
indoxyl, which in the presence of oxygen spontaneously forms indigo, a chromogenic compound
which appears blue to blue violet.
Procedure: The substrate disk is placed on a clean glass slide and one drop of distilled water was
added to moisten it. Then a heavy inoculums was rubbed onto the disk and it was incubated at
room temperature for up to 5 min. A positive test resulted in a blue to blue-violet color within 5
min, indicating the hydrolysis of IB by butyrate esterase.
O. Bile solubility test
Bile salts, specifically sodium deoxycholate and sodium taurocholate, have the capacity to
selectively lyse S. pneumoniae when added to actively growing bacteria in agar or broth media.
S. pneumoniae produces autolytic enzymes that account for the central depression or
umbilication characteristic of older pneumococcal colonies on agar media. The addition of bile
salts activates the autolysins and accelerates the natural lytic reactions observed with cultures of
pneumococci. The turbidity of a broth suspension visibly clears on addition of bile salts if the
organism is soluble.
Procedure: A heavy suspension of the organism (equivalent to 1 McFarland standard) was
prepared from growth on agar media in 0.5ml of phosphate buffered saline, PH 7.0. Then, 5 drops
of 10% sodium deoxycholate is added to one of the tubes (labeled ‘test’) and 5 drops of sterile
normal saline was added to other tube (labeled’control’).Both the tubes were gently agitated and
placed in a water bath at 350C for 3 hours, checking hourly for clearing.
P. Optchon Susceptibility test
Ethylhydrocupreine hydrochloride (optochin), a quinine derivative, selectively inhibits the
growth of S. pneumoniae at very low concentration (5ug/ml).Optochin may also inhibit other
viridians streptococci, but only at much higher concentrations. The test has a sensitivity of more
than 95%. Optochin is water soluble and diffuses readily into agar medium. Therefore, filter
paper impregnated with optochin is used in a disk diffusion test format to determine
susceptibility of suspected pneumococci and therby confirm their identity as such S. pneumoniae
cells surrounding the disk are lysed owing to changes in the surface tension, and a zone of
inhibition is produced.
Procedure: Using a straight wire, three to four well-isolated colonies of the suspected organism
was selected, streaked over a blood agar then an optochin disk was placed over it with firmly.
The plate was incubated at 350C for 18-24 hours in a CO2 incubator (5-10% CO2 ). If the zone of
inhibition was ≥ 14 mm around the disk, the organism was considered as S. pneumoniae.
Q.X-V factor dependence test
X-factor (hemin) and V factor (NAD) are required either singly or in combination to support the
growth of various species of Haemophilus on agar media. Filter paper disks impregnated with X
and V factors are commercially available. X and V-factors in disks, each being water-soluble
readily diffuse in agar culture media. These disks are placed on the surface of a medium deficient
in these factors, like Brain-heart infusion agar (BHIA), MHA which has been inoculated as a
lawn with the test organism. Factor requirements of the organism are then determined, after
overnight incubation, by observing the patterns of growth around the disks.
Procedure: A light suspension of the test organism in saline ( to reduce the carryover of nutrients
from chocolate agar) is made and it is plated onto BHIA. Then, X factor disk, V factor disk and
XV factor disks are placed on the medium at 4 o’clock, 8 o’clock and 12 o’clock positions
respectively. The plate is incubated in CO2 incubator at 350C for 18-24 hours. If the organism
shows the growth around XV factor disk only, then it is H. influenzae, and if around V and XV
disks, then it is H. parainfluenzae and other V-factor dependent Haemophilus spp.
Appendix-VII
Specimen collection
I. Expectorated sputum specimen collection
The patients were advised not to rinse mouth and gargle with nonsterile water prior to sputum
collection, since this could introduce contaminating microbiota.
The patient was instructed not to expectorate saliva or postnasal discharge into the container.
The specimen was collected from deep cough in a leak proof container by the patients.
Appendix-VIII
Biochemical reactions of isolated gram - negative bacteria
Key: +, >85% of strains positive
-, >85% of strains negative
±, 16-84% of the strains positive after 24-48 hrs at 36˚C
Bacterial isolates Test/Sub
Lac Mot Gas Ind VP Cit PDA Ure Lys Orn H2S
P.aeruginosa - + - - - + - - - - -
K.pneumoniae + - ++ - + + - + + - -
E.coli + + + + - - - - + ± -
Acinetobacter spp - - - - - ± - ± ± - -
(Lac- Lactose,Mot- motility,Ind-indole, VP- Voges Praskauer,Cit- citrate utilization, PDA-
phenyl alanine de-aminase, Ure- Urease, Lys- Lysine , Orn-Ornithine)
Appendix - IX
Zone Size interpretative Chart
(Based on results obtained using Mueller Hinton Agar)
Antimicrobial agent Symbol Disk
content
Resistant
(mm or less)
Intermediate
(mm)
Sensitive (mm
or more)
Penicillin G P 10 units
For Staphylococci,
Streptococcus
Other than S.
pneumoniae
28
19
----
20-27
29
28
Ampicillin/Amoxy
cillin
AMP/
AML
10μg
For gram negative
enteric organisms
13 14-16 17
For Staphylococcus 28 - 29
Haemophilus spp. 18 19-21 22
Streprococcus
Other than S.
pneumoniae18 19-25 26
Amoxycillin plus
Clavulanic acid for
gram negative enteric
organism
AMC 30μg
13 14-17 18
Oxacillin OX 1μg 12 - 13
CiprofloxacinGNB CIP 5μg 15 16-20 21
Haemophilus spp. - - 21
Co-trimoxazole SXT 1.25μg 10 11-15 16
S. pneumoniae 23.75μg 15 16-18 19
Cephalexin CL 30μg 14 15-17 18
Cefoxitin FOX 30μg 21 22
Cefotaxime CTX 30μg 14 15-22 23
Ceftazidime CAZ 30μg 14 15-17 18
Ceftriaxone CRO 30μg 13 14-20 21
Cefipime FEP 30μg 14 15-17 18
Gentamycin CN 10μg 12 13-14 15
Amikacin AK 30μg 14 15-16 17
Teicoplanin TEC 30μg 10 11-13 14
Vancomycin for VA 30μg
Staphylococci 14 15
Enterococci 16 17
Chloramphenicol C 30μg 12 13-17 18
S.pneumoniae 20 21
H. influenzae 25 26-28 29
Clindamycin DA 2μg 14 15-20 21
Erythromycin for ERY 15μg
Staphylococci 13 14-22 23
Streptococci 15 16-20 21
Piperacillin when
Testing for:
PRL 100μg
Pseudomonas spp 17 18
Gram negative ba
cteria
17 18-20 21
Piperacillin plus
tazobactum
TZP 110μg
When testing for
Pseudomonas spp.
17 18
Gram negative ba
cteria
17 18-20 21
Cefoperazone plus
sulbactum
75/30 75μg/
30μg
15 16-20 21
Imipenem IMP 10μg 13 14-15 16
Meropenem MEM 10μg 13 14-15 16
Source: Oxoid Unipath Ltd. Basingstoke, Hampsire, England.