AFB(Acid Fast Bacilli) smear staining and microscopy for detection of AFB bacteria in clinical specimen (for tuberculosis

(TB)) Introduction:AFB staining is simply one of several types of tests used to identify different bacteriae. AFB (Acid Fast Bacteria staining) is used primarily to identify bacteria in the genus Mycobacterium, such as Mycobacterium Tuberculosis. The process includes staining the specimen and then trying to wash out that stain by applying an acid. If the acid does NOT wash out the color that was applied, then the bacterium is called "acid fast".[1] The ZiehlNeelsen stain, also known as the acid-fast stain, was first described by two German doctors; Franz Ziehl (1859 to 1926), a bacteriologist and Friedrich Neelsen (1854 to 1898), a pathologist. It is a special bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. Mycobacterium tuberculosis is the most important of this group, as it is responsible for the disease called tuberculosis (TB). Other important Mycobacterium species involved in human disease are Mycobacterium Kansasii, Mycobacterium Marinum, and members of the Mycobacterium Avium complex. Acid fast organisms like Mycobacterium, contain large amounts of lipid substances within their cell walls called mycolic acids. These acids, resist staining by ordinary methods like a Gram stain. It can also be used to stain a few other bacteria like Nocardia. The reagents used are Ziehl Neelsen carbolfuchsin, acid alcohol and methylene blue. Acid-fast bacilli will be bright red after staining[2] To study the AFB staining we held a visit to Punjab Institute of Nuclear Medicines (PINUM). Punjab Institute of Nuclear Medicines (PINUM) now known as PINUM Cancer Hospital at Faisalabad is one of 14 working Nuclear Medicine centres of Pakistan Atomic Energy Commission.. PINUM is located adjacent to Allied Hospital on Jail Road, Faisalabad. PINUM is attached with Allied Hospital & Punjab Medical College. It provides diagnostic and 1

therapeutic services to the population of almost 16 million residing in Faisalabad and its surroundings. PINUM became functional in 1996. In addition to Nuclear Medicine and Nuclear Cardiology, a wide range of other services are also being provided. This institute is also involved in various academic and research activities. Basically we visit three labs in PINUM which are: 1. RadiommunoAssay 2. Pathology Laboratory 3. PCR Laboratory

1- RadioImmunoAssay:PINUM is the first institute to start Radioimmunoassay (RIA) services at Faisalabad. RIA Division 'is equipped with latest equipments. The staff is well experienced and the assay results are provided within a few days. Routinely performed tests include FT4, FT3, TSH, LH, FSH, progesterone, estrogen, prolactin, testosterone, Cortisol, PSA, AFP, CEA, Beta HCG, insulin and CA- 125.

In this lab we also study about the Gamma counterThe device known as a gamma counter measures gamma radiation in a sample. Working on the same principle as a scintillation detector, these counters use crystals which emit light when photons from the gamma rays interact with them. Samples are usually placed into test tubes, which are then placed into the machine. Gamma counters are generally used in research labs since most are not portable.

2- Pathology Laboratory:Pathology Lab. is well equipped with automated chemistry and hematology analyzers. Routine Clinical tests like blood & urine complete examination, blood sugar, urea, serum creatinine, uric acid, LFTs, cardiac enzyme and lipid profile etc. are conducted. The lab is being supervised by a consultant pathologist. Special investigation such as FNA and bone marrow are also carried out.

In this lab we also visit some instruments like, Haematology analyzer, chemistry analyzer, and electrolyte analyzer.

Electrolyte analyzer:
It employs the microprocessor technology. The working of the instrument is completely controlled by the computer program.It has the liquid crystal big screen display. The users can interact with the machine directly. The whole operating process is quite simple.It has automatic air bubble examining function. The accuracy of the examination is guaranteed. The instrument can be 24 hours power on, and will be automatically in the sleeping mode if there is no testing in 10 minutes. Sample-entering plate is optional. One plate contents 40 sample positions.

Haematology analyzer
These are used to perform complete blood counts, erythrocyte sedimentation rates (ESRs), or coagulation tests. Automated cell counters sample the blood, and quantify, classify, and describe cell populations using both electrical and optical techniques. Electrical analysis involves passing a dilute solution of the blood through an aperture across which an electrical current is flowing. The passage of cells through the current changes the impedance between the terminals (the Coulter principle).[3]

Chemistry analyzer
Automated chemistry analyzers i.e. for the environmental, pharmaceutical, agricultural, detergent, food and beverage laboratory.[4]

3- PCR Laboratory:Hepatitis Band C are spreading at an alarming rate all over the country especially in the region of Faisalabad. Keeping in view the needs of people, a PCR (Polymerase Chain Reaction) lab has been established at PINUM. Latest

sophisticated equipments have been installed and currently PCR for HBV and HCV have been started. In PCR lab we study following instruments.

Centrifuge Machine

Centrifuges machines involves removal of surface water/oil adhered to the surface of the material by centrifugal force. Centrifugal force is the force which throws the material radially out while performing a circular motion. the rotating unit, called the rotor, has fixed holes drilled at an angle (to the vertical). Test tubes are placed in these slots and the motor is spun.

Laminar Flow Cabinet

A laminar flow cabinet or laminar flow closet or tissue culture hood is a carefully enclosed bench designed to prevent contamination of semiconductor wafers, biological samples, or any particle sensitive device. Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the user. The cabinet is usually made of stainless steel with no gaps or joints where spores might collect.[5]

Gel Electrophoresis:
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size (IEF agarose, essentially size independent) and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.

Gel Visulization:

PCR Machine:

PCR provides direct detection of virus as compared to other techniques that detect antibodies such as ELISA, RIBA and IRMA etc. Because PCR is very sensitive, this analysis is possible soon after infection, which can be from several days to several

months before actual symptoms occur. The specificity of PCR based assays for infectious diseases is excellent (>98%). Genotyping for HCV has also been started which IS an important test for proper management of hypatitis-C patients.[6]

Review of literature:Tuberculosis (TB) is a global health problem. The World Health Organization estimated 9.4 million incident cases during 2009 and 1.7 million deaths. The challenge in reducing the global burden of tuberculosis is rapid diagnosis followed by appropriate treatment. Current methods of diagnosing TB are inadequate. The most widely used test, microscopic examination of sputum, lacks sensitivity and global cases detection rates remain low. It is estimated that only 63% of incident cases are detected each year. Definitive diagnosis requires isolation and culture of the bacilli which may take weeks and requires considerable laboratory infrastructure to protect against infection.[7] The Ziehl-Neelsen method is probably the best known and most frequently used procedure for staining tubercle bacilli. The method requires controlled heating for its success. However, in developing countries, such as India, where most laboratories rely mainly on spirit lamps as a source of heat, the Ziehl-Neelsen method often cannot be carried out because rectified spirit is difficult to obtain. The study describes a cold staining technique that uses the same staining solutions as the conventional Ziehl-Neelsen method. For direct smears, the correlation of results of the cold staining procedure with those of the Ziehl-Neelsen method was 97% and for concentrated smears was 99%. The method described is suitable for use in basically equipped laboratories.[8]
The Acid Fast stain is very useful method used to identify bacteria. It consists of a primary dye (carbol-fuchsin), a decolorizer (acid:alcohol) and a counter stain (methylene blue). The decolorizer is used to decolorize the bacteria that "do not like" the primary stain. The counter stain is used to stain those bacteria which were decolorized by the acid:alcohol.

Bacteria which retain the carbol-fuchsin are fuchsia-colored and are called acid fast bacteria (AFB). Non-AFB are decolorized by the acid:alcohol and are stained by the methylene blue and appear blue.[9]

Material and Methods:1. Staining solution:

Stock Solution A (stable for 6 months) L.O.C. High Suds (Amway) ............ 0.6 ml Distilled water .......................... 100 ml Stock Solution B Basic fuchsin ............................ 1 g Absolute ethyl alcohol .................. 10 ml The two solutions can be kept as stock solution and mixed before use.

2. Working Solution Mix 50ml of A with 5 ml of B.

3. acid alcohol - 3% hydrochloric acid in 95% ethyl alcohol Absolute ethyl alcohol .............. 95ml Distilled water ...................... 2 ml Concentrated hydrochloric acid ... 3 ml Make up the alcohol solution then add the concentrated acid. Use extreme care when handling concentrated acid. 4. 0.25% methylene blue in 1% acetic acid Methylene blue ........................ 0.25 g Distilled water ......................... 99 ml Acetic acid ............................. 1 ml[10]

1. Primary Staining: Add Ziehl-Neelsen carbol fuchsin to the slide for 5-10 minutes while applying heat.the heating stage is to make the color to go into the cell wall 2. Follow with a gentle wash with water to cool the slide. 3. Decolorising: Can be done with 20% sulfuric acid and Alcohol separately or with acid alcohol (which contains hydrochloric acid and ethanol). if the bacteri is acid fast it doesn't lose its color in this stage because of its high amouns of lipid 4. Wash the slide in water. 5. Counterstaining: Usually done with methylene blue or malachite green. The acid-fast bacteria retain the red color, and therefore look hot pink or red. Non acid-fast bacteria will not be red. If methylene blue is used the nonacid-fast organisms will appear blue, and if malachite green is used non-acidfast organisms will appear green.

Results:

Acid fast bacilli................................... Red Nuclei.............................................. Blue Other tissue constituents....................... Blue

Discussion:7

Acid-fastness is a physical property of some bacteria referring to their resistance to decolorization by acids during staining procedures. The high mycolic acid content of certain bacterial cell walls, like those of Mycobacteria, is responsible for the staining pattern of poor absorption followed by high retention.

Very few structures are acid fast, this makes staining for acid-fastness particularly useful in diagnosis: All Mycobacteria - M.tuberculosis, M.Leprae and atypical Mycobacterium Nocardia, Head of sperm, Bacterial spores, Cryptosporidium parvum, Isospora and Cyclospora cysts.[11]

Mycobacterial cell walls contain a waxy substance composed of mycolic acids. These are -hydroxy carboxylic acids with chain lengths of up to 90 carbon atoms. The property of acid fastness is related to the carbon chain length of the mycolic acid found in any particular species. Basic fuchsin binds to negatively charged groups in bacteria. The mycolic acid (and other cell wall lipids) present a barrier to dye entry as well as elusion (washing out with solvent) and this is partly overcome by adding a lipophilic agent to a concentrated aqueous solution of basic fuchsin and partly by heating. In this lab I learned about the third type of gram bacterias that is mycobacterium tuberculosis and method to stain that bacteria. The presence of bacterias in tb patients I also learned to identify and classify different types of bacterias I was able to observe the bacterias unde microscope In doing so, I gained useful microscopy skills such as making wet mount slides, finding the proper magnification for viewing, and drawing microscope observations with all the proper labels.