CDST_LT: Identification methods for MGIT 960 LC growth

The following observations can make tentative identification:

1.   Rate of growth: Generally, M. tuberculosis, M. bovis and, to some extent, M. Kansasii are slow growers and take a longer time to turn positive in an MGIT tube as compared to other non tuberculous mycobacteria (NTM). 

2.   Nature of turbidity: In liquid medium, M. tuberculosis appears as granular or flaky growth, while most NTM form uniform slight turbidity (except M. kansasii).

3.   Smear examination: M. tuberculosis forms typical clumps and serpentine cords, while other mycobacteria appear as loose, smaller clumps and cording single cells. M. kansasii may be difficult to differentiate as it is morphologically closer to M. tuberculosis.

4.   Lateral flow immunochromatography (Capilia TB Test): Used to differentiate M. tuberculosis from NTM. 

5.  Other methods: For complete speciation, other biochemical tests can be used.

   Resource

Mycobacteriology Laboratory Manual 

Assessment

Question 1	Answer 1	Answer 2	Answer 3	Answer 4	Correct Answer	Correct Explanation	Page id	Part of Pre-Test	Part of Post-Test
The majority of mycobacterial species grow well at 37°C ± 1ºC.	True	False			True	Most mycobacterial species grow well at 37°C ± 1ºC; however, some may require temperatures other than 37°C.		Yes	Yes
Question 2
The reagents for decontamination can be used, which were prepared one month back.	True	False			False	It is better to use freshly prepared reagents.		Yes	Yes

In liquid medium, M. tuberculosis appears as granular or flaky growth, while most NTM form uniform slight turbidity (except M. kansasii).

 M. tuberculosis forms typical clumps and serpentine cords, while other mycobacteria appear as loose, smaller clumps and cording or single cells. M. kansasii may be difficult to differentiate as it is morphologically closer to M. tuberculosis.

Resource

Mycobacteriology Laboratory Manual 

1. Assessment

Question 1	Answer 1	Answer 2	Answer 3	Answer 4	Correct Answer	Correct Explanation	Page id	Part of Pre-Test	Part of Post-Test
In a liquid medium, M. tuberculosis appears as granular or flaky growth.	True	False			True	In a liquid medium, M. tuberculosis appears as granular or flaky growth.		Yes	Yes
Question 2
M. tuberculosis forms typical clumps and serpentine cords.	True	False			True	M. tuberculosis forms typical clumps and serpentine cords.		Yes	Yes

 

Once an MGIT tube is positive by fluorescence or visual observation, prepare a smear and stain with carbol fuchsin stain. 

Procedure:

1. Use a clean slide. 

2. Mix the broth by vortexing, and then remove an aliquot using a sterile pipette. Place 1-2 drops on the slide and spread over a small area (approx. 1½ x 1 cm). 

3. Let the smear air dry.

4. Heat-fix the smear by passing it over a flame a few times or using an electric warmer at 65ºC - 70ºC for 2 hours to overnight. Do not leave the smear openly exposed to the UV light of the safety cabinet. 

5. Stain the smear with Ziehl-Neelsen.

6. Air dry, but do not blot dry.

7. Place a drop of oil on the stained and completely dried smear and screen under a low-power objective to locate stained bacteria. Then, switch to an oil immersion objective lens for detailed observation.

8. If the broth appears turbid or contaminated, irrespective of AFB smear results, subculture on a blood or chocolate agar, or TSI, to rule out the presence of contaminating bacteria. 

9. If the smear is negative for AFB and the tube does not appear to be contaminated, i.e. broth is clear, re-enter the tube into the instrument for further monitoring. Repeat AFB smears after 1-3 days.

   Resource

Mycobacteriology Laboratory Manual 

Assessment

Question 1	Answer 1	Answer 2	Answer 3	Answer 4	Correct Answer	Correct Explanation	Page id	Part of Pre-Test	Part of Post-Test
Fluorochrome stain is not recommended for LC AFB smears.	True	False			True	Fluorochrome stain is not recommended for LC AFB smears.		Yes	Yes
Question 2
M. tuberculosis forms typical clumps and serpentine cords.	True	False			True	M. tuberculosis forms typical clumps and serpentine cords.		Yes	Yes

Principle: 

This method detects MPT64 antigen specifically produced by the M. tuberculosis complex. It detects MPT64 a mycobacterial protein secreted by the cells during growth.

Testing method:

Immunochromatography is a double-antibody sandwich technique in which:

• An antibody labelled by colloidal particles, such as colloidal gold, reacts with target antigens to form an antigen-antibody complex.

• This complex migrates across a chromatographic carrier such as a filter paper.

•  The complex is captured by a second antibody ready-fixed in the middle of the chromatographic carrier.

If the target antigens are present in the test specimen, a colour reaction caused by the gold colloidal particles is observed at the site on the chromatographic carrier where the second antibody is fixed, and the specimen is interpreted as positive.

This kit employs the colloidal gold-labelled MPT64 monoclonal (mouse) antibody in the main reaction system. The results are visually identified as a specific antigen-antibody reaction between the monoclonal antibody and MPT64 antigens secreted by the isolate. 

However, if the growth of the M. tuberculosis complex is slight and the MPT64 concentration in the test specimen is below the detection limit, the complex may not be detected.

Resource

Mycobacteriology Laboratory Manual

Assessment

Question 1	Answer 1	Answer 2	Answer 3	Answer 4	Correct Answer	Correct Explanation	Page id	Part of Pre-Test	Part of Post-Test
Rapid antigen detection test is based on MPT64   Antigen.	True	False			True	This method detects the MPT64 antigen specifically produced by the M. tuberculosis complex.		Yes	Yes
Question 2
The kit can detect less mycobacterium growth too.	True	False			False	if the growth of the M. tuberculosis complex is slight and the MPT64 concentration in the test specimen is below the detection limit, the complex may not be detected.		Yes	Yes

This method detects the MPT64 antigen specifically produced by the M. tuberculosis complex. It detects MPT64 (a mycobacterial protein fraction from BCG), a protein secreted by the M. tuberculosis cells during growth.

The kit is a test plate that consists of a carrier strip composed of: 

• a specimen placing area

• a reagent area containing a colloidal gold-labelled anti-MPT64 monoclonal antibody (mouse)

• a developing area where the anti-MPT64 monoclonal antibody (mouse) and an anti-mouse immunoglobulin polyclonal antibody (rabbit) is fixed

A drop of the culture is put on the “specimen placing area” on the test plate. The colloidal gold labelled MPT64 antibody “A” dissolves and forms an immune complex with MPT64 antigens in the specimen. This immune complex migrates through the developing area by capillary action and is captured by the anti-MPT64 antibody “B” fixed in the reading area [T =Test]. The resultant complex forms a purple-red line of colloidal gold in the reading area [T]. This visually indicates the existence of MPT64 antigens in the specimen. On the other hand, whether or not MPT64 antigens exist in the specimen, excess colloidal gold labelled anti-MPT64 antibodies further migrate through the developing area and are captured by the anti-mouse immunoglobulin antibody (fixed antibody). The resultant complex forms a purple-red line of colloidal gold in the reading area [C = Control]. This means that the colloidal gold-labelled anti-MPT64 antibodies have migrated normally.

Precautions:

1. Use freshly prepared suspension for each quality control test. 

2. All work should be carried out in a proper biological safety cabinet.

3. All materials should be sterilized by autoclaving before disposal. 

4. Follow all the recommended safety precautions.

Resource

Mycobacteriology Laboratory Manual 

Assessment

Question 1	Answer 1	Answer 2	Answer 3	Answer 4	Correct Answer	Correct Explanation	Page id	Part of Pre-Test	Part of Post-Test
The kit is a test plate with only a reading area T.	True	False			False	The kit is a test plate consisting of a reading area, T, and a control area, C.		Yes	Yes
Question 2
The kit contains a developing area, reading area and specimen placing area.	True	True			True	The kit contains three areas - the developing areas, the reading area and the specimen placing area.		Yes	Yes

 

Specimen preparation and subsequent steps must be performed in a BSC using full PPE. 

From positive MGIT tubes:

1. Ideally, test AFB smear-positive MGIT tubes within 5 days of instrument positivity. 

2.  Vortex the tightly capped MGIT tube for 30 seconds to ensure the suspension is well-mixed.

From positive LJ slants:

1.  Test 2 to 4-week-old growth. 

2.  Add 200μL of TBC ID extraction buffer or Capilia extraction buffer to a sterile cryovial. 

3.  Using a sterile 10 μL loop, scrape a loopful of several colonies and mix with buffer, avoiding any solid medium and/or contaminants present. 

4.  Vortex the cryovial for 30 seconds to create a uniform suspension.

Further steps are the same for both :

1.  Place 100μL of the specimen, either MGIT culture or bacterial suspension from LJ slant, into the specimen well of the test device. Change pipette tips between specimens. 

2.  Start timer for 15 minutes.

3.  Examine the reading area of the test device after 15 minutes and record the test results. Do not interpret the test after 60 minutes.

   Resource

   GLI Mycobacteriology Manual

   Assessment

Question 1	Answer 1	Answer 2	Answer 3	Answer 4	Correct Answer	Correct Explanation	Page id	Part of Pre-Test	Part of Post-Test
The tightly capped MGIT tube for 30 seconds should be vortexed before performing the test.	True	False			True	The tightly capped MGIT tube must be vortexed for 30 seconds to ensure the suspension is well-mixed.		Yes	Yes
Question 2
The reading of the immunochromatographic test should not be taken after 60 min.	True	False			True	Do not interpret the test after 60 minutes.		Yes	Yes

The SD Bioline is a solid-phase immunochromatographic assay that is inexpensive, easy to use and readily available. They are easily stored at room temperature and allow the results within 15 -20 minutes.

Interpretation of Result: 

 

1) If a purple-red line appears in the Test area [T] and the control area [C], then the culture/strain is positive for the presence of the MPT64 antigens. 

 

2) If a purple-red line appears in the Control area [C] but not in the Test area [T], then the culture/strain is negative for the presence of the MPT64 antigens. 

 

3) If a purple-red line appears only in the Test area [T] and not in the Control area [C], then the test is invalid.

 

Resource

Mycobacteriology Laboratory Manual 

Assessment

Question 1	Answer 1	Answer 2	Answer 3	Answer 4	Correct Answer	Correct Explanation	Page id	Part of Pre-Test	Part of Post-Test
If a purple-red line appears only in the Test area [T] and not in the Control area [C], then the test is invalid.	True	False			True	If a purple-red line appears only in the Test area [T] and not in the Control area [C], then the test is invalid.		Yes	Yes
Question 2
If a purple-red line appears in the Control area [C] and in the Test area [T], then the culture/strain is negative for the presence of the MPT64 antigens.	True	True			False	For a culture/strain to be negative for the presence of the MPT64 antigen, a purple-red line should appear in the Control area [C] but not in the Test area [T].		Yes	Yes

 

A positive and a negative control must be tested with each new lot or a new shipment of kits received and with each new batch of extraction buffer prepared. Similarly, these controls must be run weekly or along with each batch of patient isolates when tests are set up less frequently.

1. Frequency:

• Each new lot or shipment of kits and each new prepared lot of extraction buffer. 

• Weekly, or with each batch of patient tests, if testing is performed less frequently.

2. Controls:

• Internal reagent control in the device

• Positive control: Culture of M. tuberculosis reference strain (H37Rv or H37Ra) in MGIT broth

• Negative control: Culture of a MOTT strain (e.g., a well-characterised strain of M. avium complex) in MGIT broth or broth from an uninoculated MGIT tube

3. Acceptable results: Correct results as expected for all controls

• The internal control line is visible. 

• M. tuberculosis must result in a positive test.

• MOTT strain or uninoculated broth must result in a negative test.

 4. Corrective actions: If any control result is unacceptable, do not report patient tests. 

• Repeat the test with new controls; if acceptable, repeat patient tests

• If repeat results are still unacceptable, notify the supervisor immediately and investigate potential causes for failure.

• After the investigation is complete and QC is acceptable, repeat patient tests and report results.

  Resource

   GLI Mycobacteriology Manual

1.   Assessment

Question 1	Answer 1	Answer 2	Answer 3	Answer 4	Correct Answer	Correct Explanation	Page id	Part of Pre-Test	Part of Post-Test
Each new shipment of kits and each newly prepared lot of extraction buffer should be tested.	True	False			True	Each new lot or shipment of kits and each newly prepared lot of extraction buffer should be tested.		Yes	Yes
Question 2
A culture of M. tuberculosis reference strain (H37Rv or H37Ra) in MGIT broth is present in the positive control used for immunochromatography assay.	True	False			True	The positive control used for immunochromatography assay includes a Culture of M. tuberculosis reference strain (H37Rv or H37Ra) in MGIT broth.		Yes	Yes

Biochemical Tests for MTB

An experienced laboratory technologist may make the presumptive diagnosis of tuberculosis on the basis of the morphological characteristics of tubercle bacilli, but it is best to do confirmatory tests.

Unfortunately, there is no completely reliable single test that will differentiate M. tuberculosis from other mycobacteria. Nevertheless, the following tests, when used in combination with the characteristics, will enable the precise identification of > 95% of M.tuberculosis strains:

 1) Susceptibility to p-nitrobenzoic acid (PNB)

 2) Niacin test

 3) Catalase activity at 680C/pH 7. 

Of these, the PNB test can be included along with the drug susceptibility test.

Resource

Revised National TB Control Programme Training Manual for Mycobacterium tuberculosis Culture & Drug susceptibility testing.

Assessment

Question 1	Answer 1	Answer 2	Answer 3	Answer 4	Correct Answer	Correct Explanation	Page id	Part of Pre-Test	Part of Post-Test
Biochemical tests differentiate M. tuberculosis from other mycobacteria.	True	False			True	PNB, Niacin and Catalase tests are used to differentiate M. tuberculosis from other mycobacteria.		Yes	Yes
Question 2
PNB test can be included along with the drug susceptibility test.	True	False			True	PNB test can be included along with the drug susceptibility test.		Yes	Yes

PNB p-Nitrobenzoic acid susceptibility of MTB

Principle:

The inability to grow in the presence of PNB is one of the key elements in the phenotypic differentiation of tubercle bacilli from other mycobacterial species and is part of the identification process for M. tuberculosis.

M. tuberculosis and other tubercle bacilli will not grow on culture medium containing PNB, 500 µg/ml; other mycobacterial species, with the exception of  M.gastri and some strains of M. kansasii and M. marinum, will grow in the presence of PNB.

The test must be carried out on pure cultures; otherwise, it will yield false results.

Procedure:

•      Weigh out 0.5 gm PNB and dissolve in the minimum amount of dimethylformamide (~15ml).
•       Add to 1 litre of L-J fluid, distribute and inspissate once for 50 minutes at 85 degrees. 
•       Store in a cold room.
•       Inoculate with the neat bacterial suspension one slope of LJ medium and one slope of p-nitrobenzioc acid (PNB) at a concentration of 500 µg/ml and incubate at 37⁰C for each set. 
•      Read on the 28th day.
•      PNB should not be kept for reading on the 42nd day. 
•      It is critical to inoculate with a neat suspension prepared for DST, and reading should be only on the 28th day. 

Results and Interpretation:

 M. tuberculosis does not grow on the PNB medium. All other mycobacteria are resistant to PNB.

Resource

Revised National TB Control Programme Training Manual for Mycobacterium tuberculosis Culture & Drug susceptibility testing

Assessment

Question 1	Answer 1	Answer 2	Answer 3	Answer 4	Correct Answer	Correct Explanation	Page id	Part of Pre-Test	Part of Post-Test
The reading of PNB should be taken on the 28th day.	True	False			True	The reading of PNB should be taken on the 28th day		Yes	Yes
Question 2
M.tuberculosis does not grow on the PNB medium.	True	False			True	M. tuberculosis does not grow on the PNB medium. All other mycobacteria are resistant to PNB.		Yes	Yes

Principle:

Although all mycobacteria produce niacin, comparative studies have shown that M. tuberculosis accumulates the largest amount of nicotinic acid, and its detection is useful for its definitive diagnosis. Niacin-negative M .tuberculosis strains are very rare, while very few other mycobacterial species yield positive niacin tests. 

Cultures grown on egg medium containing Asparagine yield the most consistent results in the niacin test, and LJ medium is therefore recommended. A culture must be at least three to four weeks old and must have sufficient growth of at least 100 colonies. 

Controls:

Check the reagents by testing extract from an uninoculated tube of medium (negative control) and use an extract from a culture of M. tuberculosis H37Rv as a positive control.

Reagents: 

1) O-tolidine - 1.5%: Mix 1.5 gm of O-tolidine in 100 ml of ethanol. Keep in an amber bottle and store in the dark in the refrigerator. Prepare fresh weekly.

 2) Cyanogen bromide solution, approx. 10%.: Prepare a saturated aqueous solution of cyanogen bromide (approx. 10%). Store at 4^oC in the refrigerator.

Procedure:

Results and Interpretation:

Pink colour                 = positive result

White precipitation     = negative result

Add 2-3 ml of 4%NaOH to all test tubes and discard them.

 

Precautions: 

• Cyanogen bromide is a severe lachrymator and toxic if inhaled. Work in a well-ventilated fume hood when preparing the solution and in a biological safety cabinet while testing cultures. Alternatively, commercially available niacin strips could also be used. 

• In acid solutions, cyanogen bromide hydrolyses to hydrocyanic acid, which is extremely toxic. Discard all reaction tubes into a disinfectant solution made alkaline by the addition of sodium hydroxide.

Resource

Revised National TB Control Programme Training Manual for Mycobacterium tuberculosis Culture & Drug susceptibility testing

Assessment

Question 1	Answer 1	Answer 2	Answer 3	Answer 4	Correct Answer	Correct Explanation	Page id	Part of Pre-Test	Part of Post-Test
Niacin negative M.tuberculosis  strains are very rare.	True	False			True	M. tuberculosis accumulates the largest amount of nicotinic acid, and its detection is useful for its definitive diagnosis.		Yes	Yes
Question 2
Cyanogen bromide is a severe lachrymator and toxic.	True	False			True	Cyanogen bromide is a severe lachrymator and toxic if inhaled. Therefore, work in a well-ventilated fume hood when preparing the solution and in a biological safety cabinet while testing cultures.		Yes	Yes

Principle:

The nitrate reduction test, which measures enzyme activity, is a key element in differentiating M. tuberculosis from other tubercle bacilli.

Nitrate reduction converts nitrate into nitrite, which is detected by a colourimetric test. M. tuberculosis exhibits strong nitrate reduction activity, whereas M. bovis gives a negative or weak reaction; M. Africanum strains are usually negative, but approximately 20% of strains give a positive test reaction.

The test must be carried out on pure cultures otherwise it will yield false results.

Reagents and Solutions:

Note: Discard any reagent if the colour changes or a precipitate forms.

1. Sodium nitrate substrate in buffer, pH 7

Sodium nitrate (NaNO₃)                                                                          0.085 g

Potassium dihydrogen phosphate (KH₂PO₄)                                          0.117 g

Disodium hydrogen phosphate dodecahydrate (Na₂HPO₄.12H₂O)                        0.485 g

Distilled water                                                                                          100 ml

Check pH using a pH meter. Then, sterilisee by autoclaving at 121°C for 15 minutes. 

2 ml aliquots of the substrate solution are aseptically dispensed into sterile screw-cap tubes when needed.

2.  Reagent 1: Hydrochloric acid solution

Concentrated hydrochloric acid (HCl, 36%)                                            10 ml

Distilled water                                                                                          10 ml

Add concentrated HCI slowly to distilled water.

3.  Reagent 2: Sulfanilamide solution, 0.2%

Sulfanilamide  (C₆H₈N₂O₂S)                                                                   0.2 g            

Distilled water                                                                                          100 ml

Dissolve sulfanilamide in distilled water and store it in an amber bottle in the dark in a refrigerator.

4.  Reagent 3: N-naphthylethylene diamine solution, 0.1%

N-naphthylethylene diamine dihydrochloride                                          0.1 g

Distilled water                                                                                          100 ml

Dissolve N-naphthylethylene diamine in distilled water.

Store in an amber bottle in the dark in a refrigerator.

Procedure:

The entire procedure must be carried out in a biological safety cabinet.

1. Add 0.2 ml of distilled water to a 16 x 100 mm screw-cap tube.

2. Take 2 loopful of colonies from positive culture and emulsify in water. 

3. Add 2.0 ml of NaNO₃ substrate to the tube and mix well.

4. Place in the water bath at 37 °C for 2 hours.

5. Remove the tube from the water bath.

6. Add 1 drop of reagent 1, 2 drops of reagent 2, and 2 drops of reagent 3.

7. Examine immediately for the development of a pink to red colour.

Result and interpretation:

Pink colour  = positive result

No colour     = negative result

Add a small amount of zinc powder to all negative tests:

• If nitrate is still present, it will be reduced by the zinc powder, and the colour will turn red (true negative).

• means that Thethe original reaction was posit if no colour developsive, but nitrate was reduced beyond nitrite.

  Resource

  Standard Operating Procedures (Nitrate Reduction Test).

  Assessment

Question 1	Answer 1	Answer 2	Answer 3	Answer 4	Correct Answer	Correct Explanation	Page id	Part of Pre-Test	Part of Post-Test
M. tuberculosis exhibits strong nitrate reduction activity.	True	False			True	A strong nitrate reduction activity is a key element in differentiating, a M. tuberculosis from other tubercle bacilli.		Yes	Yes
Question 2
Development of pink colour in Nitrate Reduction Test indicates a positive reaction.	True	False			True	The development of pink colour in Nitrate Reduction Test indicates the positive reactionTest.		Yes	Yes

 

Catalase Peroxidase test for MTB

Principle:

Catalase is an intracellular, soluble enzyme that splits hydrogen peroxide into water and oxygen. The oxygen bubbles into the reaction mixture to indicate catalase activity. Virtually all mycobacteria possess catalase enzymes, except for certain isoniazid–resistant mutants of M. tuberculosis and M.bovis.

Mycobacteria possess several kinds of catalase that vary in heat stability. Quantitative differences in catalase activity can be demonstrated by the 68⁰C test at pH 7 (indicates loss of catalase activity due to heat). Drug susceptible strains of M. tuberculosis lose catalase activity when heated to 68⁰C for 20 minutes. For these tests, cultures on LJ should be used.

Controls:

Check reagents by testing extract from an uninoculated tube of medium (negative control).

1.  1. 0.067M phosphate buffer solution, pH 7.0

Solution 1:

Na₂HPO₄, anhydrous :9.47 g 

Distilled water: 1 litre

Dissolve disodium phosphate in water to make a 0.067 M solution.

Solution 2: 

KH₂PO₄: 9.07 g 

Distilled water :1 litre 

Dissolve in water to make 0.067 M KH₂PO₄ solution.

Mix 61.1 ml of Solution 1 with 38.9 ml of Solution 2. Adjust the pH to 7. 

2. Hydrogen peroxide, 30% solution

 Store in the refrigerator.

3. Tween-80, 10% 

Tween- 80:10 ml 

Distilled water: 90 ml 

Mix Tween-80 with distilled water and autoclave at 121^o C for 10 minutes. 

Allow to cool. Store in the refrigerator.

4. Complete catalase reagent (Tween-peroxide mixture):

Immediately before use, mix equal parts of 10% Tween-80 and 30% hydrogen peroxide. 

Use 0.5 ml reagent for each strain to be tested.

Procedure:

Results and interpretation:

If bubbles appear (due to the production of oxygen gas), the bacteria are catalase positive. 

If no bubbles appear, the bacteria are catalase negative.

Resource

Revised National TB Control Programme Training Manual for Mycobacterium tuberculosis Culture & Drug susceptibility testing.

Assessment

Question 1	Answer 1	Answer 2	Answer 3	Answer 4	Correct Answer	Correct Explanation	Page id	Part of Pre-Test	Part of Post-Test
All mycobacteria have catalase enzymes.	True	False			False	All mycobacteria possess catalase enzymes, except for certain isoniazid–resistant mutants of M. tuberculosis and M.bovis.		Yes	Yes
Question 2
During the formation of bubbles, do not shake the tubes.	True	False			True	The tubes should not be shaken because Tween 80 may also form bubbles giving false positive results.		Yes	Yes