1. Lesson N10: LABORATORY DIAGNOSIS of tuberculosis
1.Scientifically methodical ground of theme
After a long decline in incidence that began before the availability of chemotherapy, tuberculosis
is making comeback. The hope for eradication tuberculosis is becaming an unlikely possibility. It
still remans the most common infectious cause of death in the world. About one-third of the
world’s population is infected with the Mycobacterium tuberculosis. Aroximately 8 million
cases of tuberculosis and three million deaths occur every year. Over 20, 000 cases of
tuberculosis were reported each year in the USA from 1980 to1993. The disease is seen most
often in areas of poverty, overcrowding, and poor hygiene. For the first time in decades, the
incidence of tuberculosis is increasing in developing countries. The major factors contributing to
this recent increase are: (1) associated of tuberculosis with HIV epidemic;(2) immigration from
countries where tuberculosis is common; (3) the transmission of tuberculosis in congregate
settings (e.g. healthcare facilities, correctional facilities, homeless shelters); (4) a deterioration a
healthcare infrastructure. Near 14% of Mycobacterium tuberculosis isolates were resistent to one
or more drugs and about 2% were multiply resistent (MRTB) meaning that they are not sensetive
to two or more of the major TB drugs. Patients were actually dying from TB even while
undergoing multildrug therapy. Actually, tuberculosis, like a slepping giant is waking.
2.Educational purpose
STUDENTS MUST KNOW:
1. Structure, staning properties and cultivation of Mycobacteria.
2. Fermentative properties and toxin production of Mycobacteria.
3. Differential signs of Mycobacteria.
4. Ecology of M. tuberculosis. Resistance. Pathogenicity for animals.
5. Epidemiology and pathogenesis of tuberculosis.
6. Methods of laboratory diagnosis of tuberculosis, value of allergic tests, main methods of
prophylaxis and treatment.
STUDENTS SHOULD BE ABLE TO:
• stain the smears by Ziehl-Nelseen’s technique;
• examine the smears under the microscope;
• value the growth of Mycobacteria on Loewenstein-Jensen nutrient media;
• create the scheme of laboratory diagnosis of tuberculosis.
• select preparation for diagnosis and prophylaxis of tuberculosis.
3.Chart of topic content.
Causative organisms of tuberculosis in humans and animals are Mycobacterium
tuberculosis, Mycobacterium bovis, and M. africanum.

2. Laboratory diagnosis of tuberculosis consists of bacterioscopic, bacteriological, biological,
serological, and allergological examinations.
The material to be examined includes, depending on the localization of the process: sputum,
pus, cerebrospinal fluid, faeces, and lavage waters from the stomach and bronchi. The obtained
samples are collected in sterile vessels (sputum into jars, cerebrospinal fluid and other material
into test tubes).
Bacterioscopic examination. Smears are stained with the Ziehl-Neelsen method. M.
tuberculosis stained bright red (ruby) appear as either thin, long, slightly curved or short straight
rods; occasionally, they may be characterized by granularity. M. tuberculosis are arranged singly
or in irregular groups. In staining the urine sediment destaining should be , made not only with
sulphuric acid but also with alcohol since the urine may harbour non-pathogenic acid-fast
mycobacteria of smegma (Mycobacterium smegmatis) which, unlike M. tuberculosis, are de-
stained by alcohol. If mycobacteria evade detection because of their small numbers present in
ordinary smears, this difficulty is obviated with the employment of such enriched methods as
homogenization and flotation.
homogenization (an equal volume of 1 per cent NaOH solution is added to the sputum, the
flask is tightly stoppered and shaken for 5-15minutes until the sputum is dissolved completely;
after centrifugation, the precipitate is neutralized by one or two drops of a 10 per cent
hydrochloric acid solution and smears are prepared);
flotation (the homogenized sputum is transferred into a flask which has a rubber stopper and
heated in a water bath at 55°C for 30minutes, after which it is diluted with distilled water, and 1
or 2 ml of xylol, benzine or gasoline are added; the mixture is shaken for 10minutes and after it
has been left to settle for 30 minutes, smears are made from the resulting cream-like layer).
There are other methods of sputum preparation which facilitate the demonstration of
mycobacteria.
Good results are obtained by employing luminescent microscopy with auramine and examining
the specimens under the phase-contrast microscope. M. tuberculosis fluoresce with a bright
golden-green light against a dark background.
Bacteriological examination is more effective than bacterioscopic one and makes it possible
to reveal in the examined material 20-100 and over mycobacteria per ml and also to determine
their resistance to drugs, their virulence, type, etc.
Add a double volume of 6 per cent sulphuric acid killing acid-sensitive microorganisms to the
examined material in a sterile test tube and shake the tube for 10 min. Then, centrifuge the
resultant mixture, pour off the fluid, neutralize the pellet by adding 1-2 drops of 3 per cent sodium
hydroxide or by washing it off several times with isotonic sodium chloride solution, and streak on
the appropriate medium. Faeces are treated with 4 per cent solution of sodium hydroxide, the

3. mixture is placed in an incubator for 3 hrs, centrifuged, and the residue is neutralized by 8 per
cent hydrochloric acid, after which inoculation on special media is carried out.
International Loewenstein-Jensen medium is recommended by WHO as the-standard medium
for the primary growth of M. tuberculosis and for determining their resistance to antibacterial
drugs. Dissolve 3.6 g of asparagin, 2.4 g of potassium hydrophosphate, 0.24 g of magnesium
sulphate, O.B g of magnesium citrate, and 0.4 g of potato starch and malachite green in 600 ml of
distilled water containing 12 ml of glycerol. Sterilize the mixture obtained for 15 min at 120 °C.
Then pour it into 100 ml of homogeneous suspension from fresh eggs, mix thoroughly, filter
through a cotton-gauze filter, decant into test tubes, and obtain a slant medium by coagulation at
85 °C for 45 min.
Petragnani's medium. To 150 ml of whole milk, add (with constant stirring) 6 g of potato starch,
1 g of peptone and one finely-chopped egg-sized potato. Heat tile mixture until paste is formed,
cool to 50 "C, and add four chicken eggs. and one yolk. Mix all the components, pour in 12 ml of
glycerol, and 10 ml of 2 per cent of malachite green solution, filter the mixture through a gauze
filter dispense into test tubes, and coagulate in a slant position at 85 °C for 2.5 hrs.
Glycerol potato as proposed by Pavlovsky. Peel a potato and immerse it in 1 percent solution
of mercuric chloride for 30 min, wash for 12 hrs in running water, and cut out cylinders by making
diagonal cuts. Slanted potato is placed into a Roux test tube- Pour 1 ml of 5 per cent glycerol
solution onto the bottom and sterilize the test tube.
Sauton's synthetic medium. In 200 ml of distilled water dissolve (while constantly heating) 4 g
of asparagin, 2 g of citric acid, 0.5 g of potassium dihydrophosphate, and 60 g of glycerol. Filter
the obtained mixture, supplement it with 800 ml of distilled water, add ammonium to bring pH to
7.2, decant into flasks, and sterilize for 20 min at 115 °C. To protect the mixture from drying, the
plugs. of test tubes with nutrient media are sealed with paraffin.
The composition of the Finn-2 medium is similar to that of Loewenstein-Jensen's medium, but
asparagin is replaced in it with sodium glutamate.
Samples of the cerebrospinal fluid, exudate, pus, and blood are pipetted onto a nutrient
medium without any preliminary treatment and thoroughly rubbed into it with the aid of a loop,
spreading them over the entire surface of the-medium. Cotton plugs are sealed with paraffin (to
prevent drying), the inoculated cultures are placed into a 37 °C incubator, and kept there for 6-8
weeks. An intensive growth of M. tuberculosis is observed on the 15th-25th day on the
Loewenstein-Jensen medium and on the 21th-35th day on Petragnani's medium. Colonies of M.
tuberculosis are wrinkled, dry, irregular, and protrude above the surface. If no growth is observed
within 6 weeks, make a scraping from the medium surface and examine it microscopically for the
presence of acid-fast bacteria.
To improve growth of M. tuberculosis, it is recommended that the-material examined be treated
with detergents possessing a bactericidal action (sodium laurilsulphate, rodolan, teapol,
laurosept, cetavlon, etc.) or their combination with sodium hydroxide. These-methods make it

4. possible to achieve a better homogenization of the material, to reduce the time during which
colonies form on nutrient media, and to do away with the stages of centrifugation, resuspension,
and neutralization.
If the results are negative, the study is repeated several times (at least 5), and the period of
culture inoculation is lengthened.
Rapid methods of the bacterial diagnosis of tuberculosis. The method of microcultures (Price's
method). Samples of sputum, pus. urine residue, and lavage waters are spread in a thick layer on
several sterile glass slides. Take a dried preparation with a sterile forceps and immerse it for 5
min in 6 per cent sulphuric acid, and then in a sterile isotonic sodium chloride solution to remove
acid. After that place the preparations into vials with citrate blood (add 2 ml of 5 per cent sodium
citrate to 10 ml of rabbit or sheep blood, dilute the contents in a 1:4 ratio "with distilled water, and
pour the mixture into test tubes). Put the inoculated cultures into an incubator. In 48-72 hrs the
preparation is retrieved, fixed, and then stained with the ZiehI-Neelsen method. Microcolonies in
the preparation appear as plaits which form under the impact of the lipid fraction of mycobacteria
(the cord factor); the maximal growth is observed on the 7th-10th day.
In-depth growth in haemolysed blood (Shkolnikova's method). Into tubes with citrate blood
introduce material treated with sulphuric acid and washed with isotonic sodium chloride solution.
After 6-8 days of incubation, centrifuge the medium and make smears from the pellet.
Resistance of the M. tuberculosis to drugs is determined by a serial dilution technique. For
inoculation, one may use both initial material containing no less than 5 mycobacteria per a
microscopic field (direct method) and the culture isolated from it (indirect method). WHO
recommends that the resistance of mycobacteria on Loewenstein-Jensen's medium should be
determined by adding into it, prior to coagulation, various doses of drugs.
Resistance of mycobacteria can also be determined in liquid media (with an addition of drugs
in corresponding concentrations) in which M. tuberculosis grow in a way similar to that described
by Price and Shkolnikova. At the present time the biological examination fails to find wide
employment in laboratory diagnosis since experimental animals are insensitive to the strains of
mycobacteria resistant to tubazid, phthivazid, isoniazid, and other anti-tuberculosis drugs.
Biological tests are utilized for determining the virulence of isolated M. tuberculosis which are
inoculated subcutaneously into guinea pigs with negative Mantoux's test. Two-three weeks after
inoculation one should weigh the infected guinea pig, measure its regional lymph nodes, and
make Mantoux's test which is then repeated in 6 weeks. If the results are negative, sacrifice the
animal
4 months after inoculation, examine histologically the internal organs (liver, spleen, lungs,
lymph nodes), and inoculate nutrient media. The virulence of the strain is determined by the
number of specific changes in organs (development of tubercles), changes in the expected life
span of the animal, weight loss, etc.

5. The allergy cutaneous test (Mantoux's intracutaneous test with tuberculin) is largely employed
for the determination of contamination of individuals with M. tuberculosis. The results are read in
24-48-72 hrs.
If the diameter of the infiltrate at the site of tuberculin administration does not exceed 1 mm,
the test is considered negative. If the diameter of the infiltrate is 2-4 mm, the test is doubtful, if
over
5 mm, it is positive. The tuberculin reaction may be attended with the development of
lymphangitis, regional lymphadenitis, and the appearance of vesicles or necrosis. A positive
allergic response to tuberculin administration indicates the presence of M. tuberculosis in the
body. A negative reaction in adults points to the absence of immunity to tuberculosis. As a
diagnostic test, this technique is helpful in recognizing tuberculosis in children, identifying
populations requiring revaccination against tuberculosis, and assessing the prevalence of
tuberculosis as an epidemiological indicator. Pirquet's cutaneous test that was extensively used
in the past has become outdated and is no longer utilized.
Serological diagnosis. The complement-fixation reaction is rarely employed in the diagnosis of
tuberculosis. The IHA reaction, as proposed by Middlebrook and Dubos is used more extensively.
Sensitized red blood cells (tannin-treated sheep or human 0-group erythrocytes) are utilized as
an antigen. They are mixed with an extract of M. tuberculosis or purified tuberculin (0.5 ml of
erythrocyte sediment and 10 ml of the extract), incubated for 2 hrs at 37 °C, and washed off with
centrifugation to remove excessive antigen. To run the test, the patient's serum is depleted by a
suspension of erythrocytes that have not been treated with the antigen, which excludes the
possibility of a non-specific reaction. The serum to be assayed is diluted, beginning with 1:2, 1:4,
1:8, etc. A positive reaction in a 1:8 dilution is definitely diagnostic. Positive results are recorded
in 70-90 per cent of tuberculosis patients.
To reveal antibodies, the agglutination reaction may be performed. The patient's blood serum
is diluted with isotonic sodium chloride solution in dilutions varying from 1:40 to 1:640. As an
antigen, use non-acid fast cultures of M. tuberculosis obtained as a result of penicillin action and
serologically similar to native M. tuberculosis. This reaction is extremely sensitive. It should be
remembered that even when the results of bacterioscopic and bacteriological studies are
negative, the diagnosis of tuberculosis may be based on clinical and X-ray findings.
FigN1: Classification of Main Mycobacterium Species
Species Formation of Causes
urease nicotine paracin niacin
amidase
amidase
M. + + + + Tuberculosis in humans and other
tuberculosis primates, in dogs and other animals that
were in contact with asick person

6. M. africanum + + + – Tuberculosis among inhabitants of tropical
Africa (Senegal, and other countries)
M. bovis + – + – Tuberculosis in calves, domestic and wild
animals, humans and other primates
M. kansasii + + + – Tuberculosis-like disaeses in humans,
which is marked by weak activity
M. intracellulare – + + – Severe forms of tuberculosis-like in
humans, localized lesions in pigs
M. xenopi – + + – Lesions of the lungs, urogenital system
and granuloma of the skin in humans
M. ulcerans + Ulceration of the skin in persons dwelling
in Mexico, New Guinea, Malaysia and
Africa
M. paratuberculosis Chronic diarrhoea in calves and sheep
M. microti + + – + Generalized infection in field mice
M. avium – + + – Tuberculosis in birds, some times in
calves, pigs and other animals. Infection
in humans is rare
M. leprae Leprosy in humans
M. leprae-murium Endemic affections of rats in different
parts of the world
Fig N2: Differentiation properties of M. tuberculosis and M. bovis.
Sings M. tuberculosis M. bovis
Morphology Fine, long rods Short rods
Glycerin Stimulate growth Inhibit growth
Pathogenicity for animals:
Rabbits Chronic Generalized acute
tuberculosis, less tuberculosis
sensitive then
guinea pigs
Guinea pigs Generalized Less sensitive then
tuberculosis and rabbits
death
Niacin test Positive Negative
Source of infection Humans Animals
Mechanism of transmission Air-born Alimentary

7. 4. Student’s independent study program
1. Classification of Mycobacteria.
2. Morphology, methods of staining, chemical composition, biochemical and antigenic
properties of causative agents of tuberculosis. Cultivation. Resistance i surrounding
environment.
3. Characteristic of pathogenic substances of M. tuberculosis: cord factor, lipids, endotoxin etc.
4. Pathogenicity for animals.
5. Epidemiology of tuberculosis. Pathogenesis and disease in man. Clinical findings.
6. Non sterile immunity, its features.
7. Allergic state. Allergic test and its practical value. Preparation for use.
8. Main methods of microbiological diagnosis of tuberculosis: bacterioscopic, bacteriological,
biological, serological. Enrichment methods – homogenization, flotation. Their values.
9. Nonspecific and specific prophylaxis of tuberculosis.
10. Characteristics of main mycobacteria which are causative agents of different diseases in
man. Mycobacteriosis. Laboratory diagnosis of mycobacteriosis.
5. Students’ practical activities:
1. To study the morphology of causative agents of tuberculosis in sputum smears stained with
Ziehl-Nelseen’s method.
3. To study the growth of M. tuberculosis on Loevenstein-Jensen’s medium.
4. To familiarize with media for cultivation of M. Tuberculosis (Petragnani's medium,
Loevenstein-Jensen’s, Sauton's and others)
5. To familiarize with preparation used for the allergic diagnosis, specific prophylaxis and
treatment of tuberculosis.
6. Control questions and tests
1. Microscopy of sputum from a patient with acute form of pulmonary tuberculosis is carried out
to reveal causative agent of this disease. What method of staining is the most likely to this?
A. Gram.
B. Ziehl-Neelsen.

8. C. Burri-Gins
D.Romanowsky-Giemsa
E. Neisser.
2. Diagnostic test Manteoux was carried out on a 6- year -old child, suspected of active
tubercular process. Choose an immunologic preparation, which was injected.
A. Vaccine AKDT.
B. Vaccine BCG.
C. Tuberculin.
D.Tularin
E. Vaccine Ð ﾐ DT.
3. In the smear from sputum stained by Ziehl-Neelsen method are revealed single or grouping
red acid-resistant rods. On nutrient medium the first growth has appeared in 14 days. What
species does the microorganism belong to?
A. Yersinia pseudotuberculosis.
B. Mycobacterium tuberculosis.
C. Histoplasma dubrosii.
D. Klebsiella rhinoscleromatis.
E. Coxiella burnettii.
4. The patient is 16 years old young man from countryside. The man has negative tuberculin
skin test. What should a doctor make?
A. To make BCG injection.
B. To repeat reaction in 1 month.
C. To make serologic diagnostic of tuberculosis.
D. Urgently to isolate the young man from collective.
E. To make accelerated diagnostics of tuberculosis by a Price method.

9. 5. At a maternity hospital immunization of newborn children against tuberculosis has been
conducted. What vaccine has been used?
A. Tetanus toxoid.
B. Diphtheritic toxoid.
C. BCG.
D. ТABTe.
E. DTT.
6.Vaccine against tuberculosis is:
A. Toxoid
B. Killed corpuscular
C. Split vaccine
D. Attenuated live
E. Associated
7. At school medical inspection of pupils has been conducted. The purpose was to select
children for revaccination against tuberculosis. Which one of the following tests has been used?
A. Burne test.
B. Widal test.
C. Tularin skin test.
D. Tuberculin skin test.
E. Anthraxin skin test.
8. Each of following statements concerning Mycobacterium tuberculosis is correct EXCEPT:
A. After being stained with carbolic fuchsin, Mycobacterium tuberculosis resists
decolorization with acid alcohol
B. Mycobacterium tuberculosis has a large amount of mycolic acid in the cell wall
C. Mycobacterium tuberculosis appears as a red rod in Gram-stained specimens
D. Mycobacterium tuberculosis appears as a red rod in acid-fast stained specimens
9. Each of following statements concerning Mycobacterium tuberculosis is correct EXCEPT:
A. Obligate requirement for oxygen

10. B. Niacin production
C. Acid fastness
D. Inability to grow on artificial media
E. Slow growth
10. According to respiratory type Mycobacterium tuberculosis belong to
A. Obligate anaerobes
B. Facultative anaerobes
C. Obligate aerobes
11. A positive tuberculin test indicate:
A. No immunity to Mycobacterium tuberculosis
B. Parasitic infection
C. presence of living Mycobacterium tuberculosis bacilli
C. Active pulmonary tuberculosis
D. Active non-tubercular mycobacterial infection
12.Mycobacterium tuberculosis infections elicit------------ immune response:
A. Delayed type of hypersensitivity
B. An immediate type of hypersensitivity
C. An anaphylactic
D. A humoral antibody
13.With the Ziehl-Neelsen stain, acid- fast bacilli appear----------- against a blue background:
A. Red
B. blue
C. Yellow
D. Purple
E. Pink
14. After BCG inoculation of infants, immunity to tuberculosis lasts until there are live vaccine
strains in the body. Name such kind of immunity?
A. Antitoxic
B. Passive.

11. C. Non-sterile (infection).
D. Natural
E. Sterile
15. Sputum examination on Price method revealed rod- shaped bacteria arranged in helical
cords. Which substance leads to stocking of these bacteria and serpentine- like growth?
A. Tuberculin.
B. Cord - factor.
C. Phosphatides.
D. Micolic acid.
E. РРД.
7. List of literature:
1.I. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology, Lugansk, 2004,
chapter21, p. 123-141.