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)


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)


Beef, infusion form 300.0

Casein Acid Hydrolysate 17.5

Starch 1.5

Agar 17.0


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)


Peptone 10.0

Sodium Chloride 5.0

Beef Extract 10.0

Yeast Extract 1.5

Agar 12.0


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)


Peptone 5.0

Sodium Chloride 5.0

Beef Extract 1.5

Yeast Extract 1.5


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)


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)


Buffered Peptone 7.0

Dextrose 5.0

Dipotassium Phosphate 5.0


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)


Tryptone 2.0

Sodium chloride 5.0


Bromothymol Blue 0.08

Agar 2.0


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)


Tryptone 20.0

Peptone 6.1

Ferrous ammonium sulphate 0.2

Sodium Thiosulphate 0.2

Agar 3.5


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)


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


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)


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


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)


Peptone 1.0

Dextrose 1.0

Sodium Chloride 5.0


Mono-potassium phosphate 0.8

Phenol Red 0.012

Agar 15.0


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)


Yeast extract 3

L-phenylalanine 1

Disodium hydrogen phosphate 1

Sodium chloride 5

Agar 12


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)


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


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


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


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


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


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


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


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.

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