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1
Topic:
Write a detailed examination of Tuberculosis in Microbiology Laboratory
including Zn stain & its report also mention the drugs for TB.
2
Content:
 Introduction
 Definition
 Causative Organisms
 Other Causative Organisms
 Non-Mycobacterium Genus
 How TB spreads?
 Classification
 Diagnosis of TB
 Zeihl-Neelsen Stain (Zn).
 Principle
 Difference between gram +ve & gram –ve bacteria
 Requirements of Zn staining
 Procedure of Zn staining
 Result & interpretation of Zn staining
 Reporting the sputum smear
 Auramine stain
 Principle
 Requirements
 Procedure
 Preparation of smear
 For Sputum
 For urine
 Procedure
 Result & interpretation
 Number of field to examine
 Reporting of smears
 Another test to diagnose the TB patient
 Tuberculin skin test(PPD)
 Chest X-Ray(CXR)
 Sputum Culture Test for TB
 Lowenstein-Jensen(LJ)
 Principle of LJ Medium
 Preparation Of LJ medium
 Management of TB by Drugs
 Plagiarism Screen shorts:
3
Introduction:
Tuberculosis(TB) is the one of the oldest disease known to
affect humans, is a major cause of death worldwide.
The word “Tuberculosis” is derived from Latin word.
- Round nodules / Swelling
- Condition
Definition:
Tuberculosis (TB) is a potentially fatal contagious disease that can affect any part of the
bodybut is mostly an infection of the lungs.
Causative Organisms:
Mycobacterium Tuberculosis (TB)
Mycobacterium Bovis
Tubercle
Osis
Human
Animals
4
How TB Spread?
 TB is blowout through the air from one person to another.
 The TB bacteria are put into the air when a person with TB disease of
the lungs or throat sneezes, speaks ,coughs, , or sings.
 People nearby may breathe in these bacteria & become infected.
Classification:
Diagnosis ofTB:
Zeihl-Neelsen Stain (ZN):
Principle:
Acid fast bacilli(AFB) has been ascribed to the high content & variety of lipid, fatty acid & higher alcohols
found in tubercle bacilli. A lipid particular to acid fast bacilli is a long chain mycolic acid. Acid fast is not a
property of lipid alone on integrity of the cell wall. Zn stain is modification of Ehrlich’s original method for
differential staining of acid fast bacilli by use of aniline gentian violet followed by strong nitric acid. Zn
staining technique is used because Acid fast bacilli(AFB) don’t stain well due to high lipid content.
Extra Pulmonary TB Pulmonary TB
Lymph Node TB Primary Disease
Skeletal TB Secondary Disease
Pericardial TB
Pleural TB
Bacteriological
Test
Sputum Culture Test Radiography
Tuberculin skin
test(PPD)
Zeihl-Neelsen
Stain
LJ(Lowenstein-jensen)
Chest X-
Ray(CXR)
Injection Of fluid
into the skin of the
lower arm.
Auramine stain
Liquid medium: 8-14
days
48-72 hours later
checked for a
reaction.
5
Difference betweengram +ve & gram –ve bacteria:
Requirements of Zn staining:
1. Primary stain : 0.3% Carbol Fuchsin – Dissolve 50g phenol in 100ml ethanol (90%) or
methanol(95%). Dissolve 3g basic fuchsin in the mixture & add distilled water to bring the volume to
1 L.
2. Decolorization solution : 25% Sulphuric acid
3. Counter stain: 0.3% methylene blue .It can also malachite green.
Procedure of Zn staining:
1. Make a thin smear of the material for study & heat fix by passing the slide 3-4 times through the flame
of a Bunsen burner or use a slide warmer at 65-75 C. Do not overheat.
2. Place the slide on staining rack & pour carbol fuschin over smear & heat gently underside of the slide
by passing a flame under the rack until fumes appear. Do not overheat & allow it to stand for five
minutes.
3. Wash smears with water until no color appears in the effluent.
4. Pour 20% sulphuric acid, wait for one minute & keep on repeating this step until the slide appears light
pink in color (15-20 sec).
5. Wash well with clean water.
6. Cover the smear with methylene blue or malachite green stain for one to two minutes.
7. Wash off the stain with clean water.
8. Wipe the back of the slide clean, & place it in a draining rack for the smear to air-dry (do not blot dry).
9. Observe the smear microscopically, using the 100x oil immersion objective.
Gram +ve bacteria Gram –ve bacteria
Gram reaction holds crystal violet dye & stain dark
violet or purple, they hold colored blue or purple
with gram stain when washed with absolute alcohol
& water.
Gram reaction Can be decolorized to accept counter
stain , stain red or pink, they don't hold the Gram
stain when washed with absolute alcohol &
acetone.
Peptidoglycan layer Thick (multilayered) Peptidoglycan layer Thin (single-layered)
Teichoic acids is Present Teichoic acids is Absent
Periplasmic space is Absent Periplasmic space is present
Outer membrane is absent Outer membrane is Present
Lipopolysaccharide (LPS) content is Virtually none Lipopolysaccharide (LPS) content is High
Resistance to physical disruption is high Resistance to physical disruption is low
Inhibition by basic dyes is high Inhibition by basic dyes is low
Susceptibility to anionic detergents is high Susceptibility to anionic detergents is low
Resistance to sodium azide is high Resistance to sodium azide is low
Toxins produced are Primarily Exotoxins Toxins produced are Primarily Endotoxins
6
Results & Interpretation of Zn staining:
1. Acid Fast Bacilli : Slightly curved rods or red, straight , occurring singly or in small groups, may
appear beaded
2. Cells : Green (malachite green) or Blue (methylene blue)
3. Background material : Green (malachite green) or Blue (methylene blue)
Reporting the Sputum Smear:
When any definite red bacilli are seen:
Report the smear as ‘AFB POSITIVE’, & give an indication of the number of bacteria present as follows:
Number of AFB seen (1000X Magnification) Reported As
0 AFB per 300 Field AFB Not Seen
1-2 AFB per 300 Fields Doubtful; repeat with another specimen
1-9 AFB per 100 Fields 1+
1-9 AFB per 10 Fields 2+
1-9 AFB per Field 3+
>9 AFB per Field 4+
7
Auramine stain:
Principle:
The fluorochrome dye, Auramine-Rhodamine, forms a complex with mycolic acids found in the acid-fast
cell wall of organisms which stops decolorization by acid-alcohol. The counterstain, permanganate, renders
tissue, potassium permanganate, renders tissue & its debris nonfluorescent, thus reducing the possibility of
artifacts. The cells visualized under UV light seems bright yellow.
Requirements:
1. Counter Stain: 0.5% Pottassium Permanganate (KMnO4) (0.25 gm in 50 ml).
2. Primary Stain: Auramine Rhodamine Solution.
3. Decolorizer: 0.5% Acid alcohol (5 ml hydrochloric acid in 995 ml 70% alcohol).
Procedure:
1. Preparation of Smear:
1. ForSputum:
Using a piece of stick, transfer a purulent part of the sputum to a slide & make a thin smear. An area of
approximately 2-cm square is recommended. Spread the smear using circular movements. Allow to air
dry.
2. ForUrine: Prepare a smear of the deposit from three centrifuged early morning urine
sediments.
Give permission to air dry & heat fix the sample. Use of an electrical slide warmer is commonly the
favored process for heat-fixing smears. An substitute method of heat-fixing is to pass the dried slide,
smear facing upward, two to three times through the blue cone of a burner flame.
2. Procedure:
1) Place the fixed smear on a staining rack & flood slide with rhodamine-auramine for 15 minutes. Do
not let surface dry.
2) Wash off the stain with distilled water.
3) Cover the slide with fluorescent decolorizer (i.e acid-alcohol) for 2-3 minutes.
4) Wash thoroughly with distilled water.
5) Flood slide with potassium permanganate for 3-4 minutes. Do not allow slide to dry.
6) Wash thoroughly with distilled water & air dry.
7) Observe microscopically under the same light source as used for fluorescent microscopy
8) Slides can be screened on high power (400X) & verified under oil immersion.
8
Result & Interpretation:
1) Positive Test – AFB fluoresce reddish-orange against a dark background.
2) Negative Test – Non-AFB will not fluoresce or may appear a pale yellow, quite distinct from the
bright acid-fast organisms.
Number of Fields to Examine:
The minimum number of fields to examine before reporting a smear as negative for AFB. Final
magnification (the objective lens magnification multiplied by the eyepiece magnification) Vs. Number of
slides e.g.
1. 200x: 30
2. 250x: 30
3. 400x: 55
4. 450x: 70
Reporting of smears:
 If Fluorescent AFB are seen,report the smear as AFB positive, & give an indication of the number of
bacilli present in plus signs (+ to +++)
 If no fluorescent rods are seen, report the smear as NO acid fast bacilli seen.
Report
Number of AFB observed
200x,250x 400x,450x
No AFB seen 0 0
Doubtful: Repeat 1-2/30F 1-2/70F
1+ 1-9/10F 2-18/50F
2+ 1-9/F 4-36/10F
3+ 10-90/F 4-36/F
4+ >90/F >36/F
9
Another test to diagnose the TB patient:
Tuberculin skin test(PPD):
Procedure to perform the Tuberculin skin test PPD:
1) The tuberculosis skin test is another name for the tuberculin test or purified protein derivative (PPD)
test.
2) The purified protein derivative (PPD) test checks if
patient has developed an immune response to the
bacterium that causes tuberculosis (TB).
3) The suggested tuberculin test is also known as
Mantoux test, which is managed by injecting a 0.1
mL of liquid containing five tuberculin units purified
protein derivative into the top layers of skin of the
forearm.
4) Doctors should check skin (Induration area) 48-72 hours after the injection.
5) The basis of the reading of the skin test is the presence or absence & the amount of induration
(localized swelling).
6) A negative test does not mean that a person is free of tuberculosis (TB).
7) A person who received a Bacille Calmette Guerin (BCG) vaccine against tuberculosis may also have
a positive skin reaction to the tuberculin (TB) test.
8) It is also known as mantoux test.
10
Chest X-Ray(CXR):
Tuberculosis (TB) is an infectious disease that causes swelling, the
development of tubercles & other developments within tissue, & can
cause tissue death. These chest x-rays show progressive pulmonary
tuberculosis (TB). There are several light areas (opacities) of varying
size that run together (coalesceArrows indicate the location of cavities
within these light areas). The x-ray on the left obviously shows that the
opacities are located in the upper area of the lungs toward the back. The
appearance is typical for chronic pulmonary TB but may also happen
with chronic pulmonary histiocytosis & chronic pulmonary
coccidioidomycosis. Pulmonary tuberculosis (TB) is making a
comeback with new resistant strains that are difficult to treat.
Pulmonary tuberculosis (TB) is the most common form of the disease, but other organs can be infected.
Sputum Culture Test for TB:
Lowenstein-Jensen (LJ)
Lowenstein-Jensen (LJ) is the selective medium which is used for the
cultivation & isolation of Mycobacterium species. It was developed
by Lowenstein who incorporated congo red & malachite green to inhibit
unwanted bacteria. The present preparation, a glycerated egg-based medium,
is based upon Jensen’s modification. Jensen’s version rejects congo red &
uses a moderate amount of malachite green to stop growth of the majority of
contaminants surviving decontamination of the specimen. This formulation
also encourages the earliest growth of mycobacteria.
Principle of LJ Medium:
L-Asparagine & Potato Flour are sources of nitrogen & vitamins. Monopotassium Phosphate & Magnesium
Sulfate enhance organism growth & act as buffers. Malachite green, stop the growth of the bulk of
contaminants surviving decontamination of the specimen while encouraging the development of
Mycobacteria. Egg Suspension provide fatty acids & protein required for the metabolism of
mycobacteria.. Glycerol serves as a carbon source & is favorable to the growth of the human type tubercle
bacillus while being unfavorable to the bovine type.
Preparationof LJ Medium:
1. Dissolve 37.3 gm of the medium in 600 ml of purified water having 12 ml of glycerol.
2. Heat if essential to dissolve the medium completely.
3. Autoclave at 121°C for 15 minutes.
4. Prepare 1000 ml of a uniform suspension of fresh eggs under aseptic situations. Avoid flogging
5. air into suspension during the collection & mixing.
6. Aseptically mix the 1000 ml of egg suspension with 600 ml of the sterile LJ Medium cooled to 50 –
60°C, avoiding air bubbles.
11
7. Dispense the completed medium into sterile screw-cap test tubes.
8. Place the tubes in a slanted position & heat at 85°C for 45 minutes.
Management of TB by Drugs:
Isoniazid Streptomycin
Rifampicin Ethambutol

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Detailed examination of tuberculosis in microbiology laboratory including zn stain & its report also mention the drugs for tb.

  • 1. 1 Topic: Write a detailed examination of Tuberculosis in Microbiology Laboratory including Zn stain & its report also mention the drugs for TB.
  • 2. 2 Content:  Introduction  Definition  Causative Organisms  Other Causative Organisms  Non-Mycobacterium Genus  How TB spreads?  Classification  Diagnosis of TB  Zeihl-Neelsen Stain (Zn).  Principle  Difference between gram +ve & gram –ve bacteria  Requirements of Zn staining  Procedure of Zn staining  Result & interpretation of Zn staining  Reporting the sputum smear  Auramine stain  Principle  Requirements  Procedure  Preparation of smear  For Sputum  For urine  Procedure  Result & interpretation  Number of field to examine  Reporting of smears  Another test to diagnose the TB patient  Tuberculin skin test(PPD)  Chest X-Ray(CXR)  Sputum Culture Test for TB  Lowenstein-Jensen(LJ)  Principle of LJ Medium  Preparation Of LJ medium  Management of TB by Drugs  Plagiarism Screen shorts:
  • 3. 3 Introduction: Tuberculosis(TB) is the one of the oldest disease known to affect humans, is a major cause of death worldwide. The word “Tuberculosis” is derived from Latin word. - Round nodules / Swelling - Condition Definition: Tuberculosis (TB) is a potentially fatal contagious disease that can affect any part of the bodybut is mostly an infection of the lungs. Causative Organisms: Mycobacterium Tuberculosis (TB) Mycobacterium Bovis Tubercle Osis Human Animals
  • 4. 4 How TB Spread?  TB is blowout through the air from one person to another.  The TB bacteria are put into the air when a person with TB disease of the lungs or throat sneezes, speaks ,coughs, , or sings.  People nearby may breathe in these bacteria & become infected. Classification: Diagnosis ofTB: Zeihl-Neelsen Stain (ZN): Principle: Acid fast bacilli(AFB) has been ascribed to the high content & variety of lipid, fatty acid & higher alcohols found in tubercle bacilli. A lipid particular to acid fast bacilli is a long chain mycolic acid. Acid fast is not a property of lipid alone on integrity of the cell wall. Zn stain is modification of Ehrlich’s original method for differential staining of acid fast bacilli by use of aniline gentian violet followed by strong nitric acid. Zn staining technique is used because Acid fast bacilli(AFB) don’t stain well due to high lipid content. Extra Pulmonary TB Pulmonary TB Lymph Node TB Primary Disease Skeletal TB Secondary Disease Pericardial TB Pleural TB Bacteriological Test Sputum Culture Test Radiography Tuberculin skin test(PPD) Zeihl-Neelsen Stain LJ(Lowenstein-jensen) Chest X- Ray(CXR) Injection Of fluid into the skin of the lower arm. Auramine stain Liquid medium: 8-14 days 48-72 hours later checked for a reaction.
  • 5. 5 Difference betweengram +ve & gram –ve bacteria: Requirements of Zn staining: 1. Primary stain : 0.3% Carbol Fuchsin – Dissolve 50g phenol in 100ml ethanol (90%) or methanol(95%). Dissolve 3g basic fuchsin in the mixture & add distilled water to bring the volume to 1 L. 2. Decolorization solution : 25% Sulphuric acid 3. Counter stain: 0.3% methylene blue .It can also malachite green. Procedure of Zn staining: 1. Make a thin smear of the material for study & heat fix by passing the slide 3-4 times through the flame of a Bunsen burner or use a slide warmer at 65-75 C. Do not overheat. 2. Place the slide on staining rack & pour carbol fuschin over smear & heat gently underside of the slide by passing a flame under the rack until fumes appear. Do not overheat & allow it to stand for five minutes. 3. Wash smears with water until no color appears in the effluent. 4. Pour 20% sulphuric acid, wait for one minute & keep on repeating this step until the slide appears light pink in color (15-20 sec). 5. Wash well with clean water. 6. Cover the smear with methylene blue or malachite green stain for one to two minutes. 7. Wash off the stain with clean water. 8. Wipe the back of the slide clean, & place it in a draining rack for the smear to air-dry (do not blot dry). 9. Observe the smear microscopically, using the 100x oil immersion objective. Gram +ve bacteria Gram –ve bacteria Gram reaction holds crystal violet dye & stain dark violet or purple, they hold colored blue or purple with gram stain when washed with absolute alcohol & water. Gram reaction Can be decolorized to accept counter stain , stain red or pink, they don't hold the Gram stain when washed with absolute alcohol & acetone. Peptidoglycan layer Thick (multilayered) Peptidoglycan layer Thin (single-layered) Teichoic acids is Present Teichoic acids is Absent Periplasmic space is Absent Periplasmic space is present Outer membrane is absent Outer membrane is Present Lipopolysaccharide (LPS) content is Virtually none Lipopolysaccharide (LPS) content is High Resistance to physical disruption is high Resistance to physical disruption is low Inhibition by basic dyes is high Inhibition by basic dyes is low Susceptibility to anionic detergents is high Susceptibility to anionic detergents is low Resistance to sodium azide is high Resistance to sodium azide is low Toxins produced are Primarily Exotoxins Toxins produced are Primarily Endotoxins
  • 6. 6 Results & Interpretation of Zn staining: 1. Acid Fast Bacilli : Slightly curved rods or red, straight , occurring singly or in small groups, may appear beaded 2. Cells : Green (malachite green) or Blue (methylene blue) 3. Background material : Green (malachite green) or Blue (methylene blue) Reporting the Sputum Smear: When any definite red bacilli are seen: Report the smear as ‘AFB POSITIVE’, & give an indication of the number of bacteria present as follows: Number of AFB seen (1000X Magnification) Reported As 0 AFB per 300 Field AFB Not Seen 1-2 AFB per 300 Fields Doubtful; repeat with another specimen 1-9 AFB per 100 Fields 1+ 1-9 AFB per 10 Fields 2+ 1-9 AFB per Field 3+ >9 AFB per Field 4+
  • 7. 7 Auramine stain: Principle: The fluorochrome dye, Auramine-Rhodamine, forms a complex with mycolic acids found in the acid-fast cell wall of organisms which stops decolorization by acid-alcohol. The counterstain, permanganate, renders tissue, potassium permanganate, renders tissue & its debris nonfluorescent, thus reducing the possibility of artifacts. The cells visualized under UV light seems bright yellow. Requirements: 1. Counter Stain: 0.5% Pottassium Permanganate (KMnO4) (0.25 gm in 50 ml). 2. Primary Stain: Auramine Rhodamine Solution. 3. Decolorizer: 0.5% Acid alcohol (5 ml hydrochloric acid in 995 ml 70% alcohol). Procedure: 1. Preparation of Smear: 1. ForSputum: Using a piece of stick, transfer a purulent part of the sputum to a slide & make a thin smear. An area of approximately 2-cm square is recommended. Spread the smear using circular movements. Allow to air dry. 2. ForUrine: Prepare a smear of the deposit from three centrifuged early morning urine sediments. Give permission to air dry & heat fix the sample. Use of an electrical slide warmer is commonly the favored process for heat-fixing smears. An substitute method of heat-fixing is to pass the dried slide, smear facing upward, two to three times through the blue cone of a burner flame. 2. Procedure: 1) Place the fixed smear on a staining rack & flood slide with rhodamine-auramine for 15 minutes. Do not let surface dry. 2) Wash off the stain with distilled water. 3) Cover the slide with fluorescent decolorizer (i.e acid-alcohol) for 2-3 minutes. 4) Wash thoroughly with distilled water. 5) Flood slide with potassium permanganate for 3-4 minutes. Do not allow slide to dry. 6) Wash thoroughly with distilled water & air dry. 7) Observe microscopically under the same light source as used for fluorescent microscopy 8) Slides can be screened on high power (400X) & verified under oil immersion.
  • 8. 8 Result & Interpretation: 1) Positive Test – AFB fluoresce reddish-orange against a dark background. 2) Negative Test – Non-AFB will not fluoresce or may appear a pale yellow, quite distinct from the bright acid-fast organisms. Number of Fields to Examine: The minimum number of fields to examine before reporting a smear as negative for AFB. Final magnification (the objective lens magnification multiplied by the eyepiece magnification) Vs. Number of slides e.g. 1. 200x: 30 2. 250x: 30 3. 400x: 55 4. 450x: 70 Reporting of smears:  If Fluorescent AFB are seen,report the smear as AFB positive, & give an indication of the number of bacilli present in plus signs (+ to +++)  If no fluorescent rods are seen, report the smear as NO acid fast bacilli seen. Report Number of AFB observed 200x,250x 400x,450x No AFB seen 0 0 Doubtful: Repeat 1-2/30F 1-2/70F 1+ 1-9/10F 2-18/50F 2+ 1-9/F 4-36/10F 3+ 10-90/F 4-36/F 4+ >90/F >36/F
  • 9. 9 Another test to diagnose the TB patient: Tuberculin skin test(PPD): Procedure to perform the Tuberculin skin test PPD: 1) The tuberculosis skin test is another name for the tuberculin test or purified protein derivative (PPD) test. 2) The purified protein derivative (PPD) test checks if patient has developed an immune response to the bacterium that causes tuberculosis (TB). 3) The suggested tuberculin test is also known as Mantoux test, which is managed by injecting a 0.1 mL of liquid containing five tuberculin units purified protein derivative into the top layers of skin of the forearm. 4) Doctors should check skin (Induration area) 48-72 hours after the injection. 5) The basis of the reading of the skin test is the presence or absence & the amount of induration (localized swelling). 6) A negative test does not mean that a person is free of tuberculosis (TB). 7) A person who received a Bacille Calmette Guerin (BCG) vaccine against tuberculosis may also have a positive skin reaction to the tuberculin (TB) test. 8) It is also known as mantoux test.
  • 10. 10 Chest X-Ray(CXR): Tuberculosis (TB) is an infectious disease that causes swelling, the development of tubercles & other developments within tissue, & can cause tissue death. These chest x-rays show progressive pulmonary tuberculosis (TB). There are several light areas (opacities) of varying size that run together (coalesceArrows indicate the location of cavities within these light areas). The x-ray on the left obviously shows that the opacities are located in the upper area of the lungs toward the back. The appearance is typical for chronic pulmonary TB but may also happen with chronic pulmonary histiocytosis & chronic pulmonary coccidioidomycosis. Pulmonary tuberculosis (TB) is making a comeback with new resistant strains that are difficult to treat. Pulmonary tuberculosis (TB) is the most common form of the disease, but other organs can be infected. Sputum Culture Test for TB: Lowenstein-Jensen (LJ) Lowenstein-Jensen (LJ) is the selective medium which is used for the cultivation & isolation of Mycobacterium species. It was developed by Lowenstein who incorporated congo red & malachite green to inhibit unwanted bacteria. The present preparation, a glycerated egg-based medium, is based upon Jensen’s modification. Jensen’s version rejects congo red & uses a moderate amount of malachite green to stop growth of the majority of contaminants surviving decontamination of the specimen. This formulation also encourages the earliest growth of mycobacteria. Principle of LJ Medium: L-Asparagine & Potato Flour are sources of nitrogen & vitamins. Monopotassium Phosphate & Magnesium Sulfate enhance organism growth & act as buffers. Malachite green, stop the growth of the bulk of contaminants surviving decontamination of the specimen while encouraging the development of Mycobacteria. Egg Suspension provide fatty acids & protein required for the metabolism of mycobacteria.. Glycerol serves as a carbon source & is favorable to the growth of the human type tubercle bacillus while being unfavorable to the bovine type. Preparationof LJ Medium: 1. Dissolve 37.3 gm of the medium in 600 ml of purified water having 12 ml of glycerol. 2. Heat if essential to dissolve the medium completely. 3. Autoclave at 121°C for 15 minutes. 4. Prepare 1000 ml of a uniform suspension of fresh eggs under aseptic situations. Avoid flogging 5. air into suspension during the collection & mixing. 6. Aseptically mix the 1000 ml of egg suspension with 600 ml of the sterile LJ Medium cooled to 50 – 60°C, avoiding air bubbles.
  • 11. 11 7. Dispense the completed medium into sterile screw-cap test tubes. 8. Place the tubes in a slanted position & heat at 85°C for 45 minutes. Management of TB by Drugs: Isoniazid Streptomycin Rifampicin Ethambutol