MODULE 4 PART 2: LABORATORY DIAGNOSIS TO IDENTIFY Mycobacteria spp.

Specimen that are found within the body that we expect to NOT have any microbes

Sterile Specimen

These are the examples of sterile specimen

Blood, CSF, Synovial Fluid, Biopsy from deep organs

What is the primary specimen for Mycobacterium?

Sputum

These are specimen that we EXCRETE

Non-sterile specimen

This specimen requires digestion and decontamination

Non sterile specimen containing mucus

This specimen only requires decontamination

Non sterile specimen without mucus

process of removing contaminants and normal flora.

decontamination

Sputum quality is determined using?

Bartlet’s Classification

ACCORDING TO BARTLET’S CLASSIFICATION. Specimen is SPUTUM if:

• PMN: _______

• SEC: _______

PMN: more than 25

SEC: less than 10

ACCORDING TO BARTLET’S CLASSIFICATION. Specimen is SALIVA if:

• PMN: _______

• SEC: _______

PMN: less than 25

SEC: more than 10

process of liquefying the sample (mucus)

DIGESTION

Process of removing the contaminants

DECONTAMINATION

These methods are preferred for specimen containing Pseudomonas spp./Pseudomonas aeruginosa

Zephiran-Trisodium Phosphate Method and 5-6% Oxalic acid Method

Reagent used in Sodium Hydroxide Method?

4% Sodium Hydroxide

Sodium Hydroxide Method is also known as

Modified Petroff Method

utilize to decontaminate thin (malalabnaw) specimen such as urine

Sulfuric Acid Method

Ideal for transport of specimen

1% Cetylpyridium Chloride + 2% NaOH

1% Cetylpyridium Chloride + 2% NaOH can prolong shelf-life of sputum for up to _____?

8 days

Dimensions of smear preparation

2×3cm

Completely airdrying the smear is important so that we can prevent what?

Aerolization

When preparing the smear for AFS, where must the sputum be dislodged?

sand containing 70% alcohol.

Methods used for AFS

Ziehl Neelsen and Kinyoun

Fluorescent stain used in AFS

Auramine-Rhodamine

Acid fast Microscopy Reporting is based on?

A. National Tuberculosis Control Program Manual of Procedures by the DOH

B. Microscope used

C. Both A and B

D. None of these

C

When the reporting grade is 0, what are the values for:

• Brightfield Microscope (x1000): ____

• 200x magnification ____

• 400x magnification ____

No AFB/300 fields; No AFB/1 length; No AFB/1 length

If the result is 1-4 AFB/1 length OR 1-2 AFB/1 length, what do we need to do?

Perform confirmation test by recollecting another sample and repeating the count

1 length: one end to the other end is equivalent to ___ fields. (200x magnification)

30

1 length: one end to the other end is equivalent to ___ fields. (400x magnification)

40

After microscopy and is positive for AFB, what do we need to perform?

culture

When the reporting grade is +n, what are the values for:

• Brightfield Microscope (x1000): ____

• 200x magnification ____

• 400x magnification ____

1-9 AFB/100 field; 5-49 AFB/1 length; 3-24 AFB/1 length

When the reporting grade is 1+, what are the values for:

• Brightfield Microscope (x1000): ____

• 200x magnification ____

• 400x magnification ____

10-99 AFB/100 field; 3-24 AFB/1 field; 1-6 AFB/1 field

When the reporting grade is 2+, what are the values for:

• Brightfield Microscope (x1000): ____

• 200x magnification ____

• 400x magnification ____

1-10 AFB/field, at least 50 fields; 25-250 AFB/1 field; 7-60 AFB/1 field

When the reporting grade is 3+, what are the values for:

• Brightfield Microscope (x1000): ____

• 200x magnification ____

• 400x magnification ___

>10 AFB/field, at least 20 fields; >250 AFB/1 field; >60 AFB/1 field

How do you know if you have achieved 300 fields using the Brightfield Microscope?

Pass through the smear (with 2x3cm dimension) on each ends, top and bottom. (EACH containing 150 fields)

All are properties of EGG BASED Media except for?

A. Has whole egg

B. Has malachite green

C. Can be Selective and Non-selective

D. None of these

D

Most commonly used egg-based media and what is the amount of malachite green present?

Lowenstein Jensen Media; 0.025%

This is more inhibitory than LJ media because it contains higher concentrations of malachite green.

Also include the amounts of malachite green present.

Petragnani; 0.052%

Non selective egg based media that is recommended for heavily contaminated specimens

Petragnani

Less inhibitory because it has lower concentrations of malachite green.

Also include the amounts of malachite green present in this media

American Thoracic Society (ATS); 0.02%

Non selective egg based media that promotes growth of Mycobacterium avium complex

Wallenstein Media

Modified LJ Media

Gruft

In Gruft Media, in addition to malachite green it contains ______ and ______ as additional inhibitors

penicillin, nalidixic acid

In Mycobactosel LJ media, in addition to malachite green it contains _____, ______, and ______ as inhibitors

cycloheximide, lincomycin, nalidixic acid

Protein source for agar based media

Serum albumin

This media contains salts, vitamins, cofactors, oleic acid, albumin, catalase, glycerol, and malachite green

Middlebrooke Media

Purpose of oleic acid in Middlebrooke Media

For metabolism of Mycobacteria

Purpose of albumin in Middlebrooke media

protects AFB against toxic agents

Agar based media that contains glucose and dextrans

Middlebrooke 7H10

Middlebrooke 7H11 is 7H10 with _______

0.1% casein hydrolysate

Middlebrooke 7H11 is used for improved recovery of?

Isoniazid-resistant Mycobacterium tuberculosis

contains cycloheximide, lincomycin, nalidixic acid in addition to malachite green

Middlebrooke 7H10 (selective)

contains carbenicillin, amphotericin B, polymyxin B, Trimethoprim lactate

Mitchison’s selective 7H11

This media is used for rapid culture

Liquid Media

What is the purpose of culturing in a liquid media?

For continuous monitoring of Mycobacterial growth

non-selective broth used to subculture stock culture

Middlebrooke 7H9 and Dubos Tween Albumin Broth

Modification of Middlebrooke 7H9

Mycobacteria Growth Indicator Tube (MGIT)

Mycobacteria Growth Indicator Tube (MGIT) contains?

fluorescence quenching-based oxygen sensor

Used in MGIT960 system

BacTec 12B

BacTec 12B has _____, that is consumed by Mycobacteria making them produce CO2, indicating positive growth of Mycobacteria

14C-labelled palmitic acid

utilized for rapid growth and identification of Mycobacteria

Septi-Chek AFB

WHEN YOU SEE AN ACID FAST BACTERIA IN THE PREPARED SMEAR/MICROSCOPY, what is the recommended culture method?

culture in one solid based and one liquid-based media

When there is positive growth in the media, what should we perform next?

Biochemical tests

Purpose of performing biochemical test

To ID the Mycobacterium spp. present in the specimen

enzyme that converts free niacin to niacin ribonucleotide

Niacin connecting enzyme

Which biochemical test has this principle?

Most Mycobacteria spp. produce niacin and most possess another enzyme that converts free niacin to niacin ribonucleotide.

Niacin Test

Niacin Test is also known as?

Nicotinic Acid/Niacin Accumulation Test

The M/C test to ID Mycobacterium tuberculosis

Niacin test

Media for Niacin test

Lowenstein Jensen Media

Reagent for Niacin Test

Strip impregnated with Cyanogen Bromide

Positive reaction for Niacin test

Yellow color or Canary Yellow

Which biochemical test has this principle?

Detects for the production of nitroreductase/nitrate reductase by bacteria that reduce nitrate to nitrite

Nitrate Reduction Test

Media for Nitrate Reduction Test

Sodium nitrate both

Reagents for Nitrate reduction test

50% HCl, Sulfanilamide , N-naphthylenediamine dihydrochloride , Zinc

Positive reaction for Nitrate Reduction Test

Pink to red color

If there is a negative reaction for nitrate reduction test, what do we need to do?

add zinc powder

True negative reaction for nitrate reduction test is indicated by?

Pink to red color caused by the reduction of nitrate to nitrite by the zinc dust

In Nitrate reduction test, once we added zinc dust as our confirmation test, and the solution remains colorless, is the result positive or negative, and why?

Positive, because the bacteria has further reduced nitrate to nitrous oxide

Reaction for grading Mycobacteria: FAINT PINK

Trace

Reaction for grading Mycobacteria: CLEAR PINK

1+

Reaction for grading Mycobacteria: DEEP PINK

2+

Reaction for grading Mycobacteria: RED

3+

Reaction for grading Mycobacteria: DEEP RED

4+

Reaction for grading Mycobacteria: PURPLISH RED

5+

Which Mycobacterium spp. are expected to have a POSITIVE reaction in Nitrate Reduction Test?

Mycobacterium kansasii, Mycobacterium szulgai, Mycobacterium terrae complex, Mycobacterium tuberculosis, and Rapid growers (except Mycobacterium chelonae)

Which biochemical reaction has this principle?

Detection of arylsulfatase enzyme produced by bacteria

Arysulfatase Test

This enzyme converts tripotassium phenolphthalein to free phenolphthalein

Arylsulfatase

Reagent for arylsulfatase

Tripotassium phenolphthalein

Positive reaction for arylsulfatase test

Pink color

Which spp of Mycobacteria hydrolyzes Tripotassium phenolphthalein to free phenolphthalein after 12 days?

M. marinum, M. szulgai

Which spp of Mycobacteria hydrolyzes Tripotassium phenolphthalein to free phenolphthalein after 3 days?

M. fortuitum complex, M. chelonae, M. xenopi, M. triviale

This test detects the ability of some Mycobacteria to convert ferric ammonium citrate to iron oxide

Iron Uptake Test

Used for differentiating M. chelonae from other rapid growers.

Iron Uptake Test

How long do we incubate for iron uptake test

2 weeks

Media used for Iron Uptake Test

Lowenstein Jensen Media

Reagent for iron uptake test

20% ferric ammonium citrate

Positive reaction for iron uptake test

Rusty brown colonies

Which Mycobacterium spp. shows a POSITIVE reaction in Iron uptake test?

All rapid growers except M. chelonae.

This test detects heat labile catalase such as Mycobacterium tuberculosis catalase

Heat Stable Catalase

Media for HSC

Tween 80

Reagent for HSC

30% H2O2