Study of mycobacteria

 Mycobacterium

Classification of bacteria :-   it is classified in to six groups:-

A.      Tubercle Bacilli  

Sn.	Bacilli	eg
1	HUMAN TYPE	Mycobacvterium tuberculosis.
2	Bovin type	Mycobacterium bovin
3	Myrin type	Mycobacterium mycrotti
4	Avian type	Mycobacterrium Avian
5	Cold Blooded type	Mycobacterium mariumun

 

B.     Lepra Bacillus

Sn.	Bacillus	eg
1	Human type	Mycobacterium Leprae
2	Rat type	Mycrobacterium Lapraemurium

 

C.      Jobenas Bacillus

1.       Mycobacterium paratuberculosis also known as mycobacterium johner.

D.      Atypical Mycobacteria

1.       Photochromogens

2.       Scotochromogens

3.       Non photochromogens

4.       Rapids grower

E.       Mycobacterium causing skin ulcer.

1.       Mycobacterium ulcerans

2.       Mycobacterium balnnei

F.       Saprophytic mycobacterium (Non-Pathogenic)

1.       Mycobacterium smegmatis (sigma bacillus)

2.        Mycobacterium butrycum (Butteer)

3.       Mycobacterium phlei (Grass)

4.       Mycobacterium stercorsis (Stool & drug beap)

Lab diagnosis of mycobacterium tuberculosis:

1.       Direct smear examination of sputum / urinary deposite / or any exudate – ZN Staining or Auramin stain for AFB.

2.       Culture from Laryngeal swab / Gastric lavage/Uri nary deposite/Endomaterial Tissue/Sputum on LJ  medium for 6 mon ths – Look for AFB

3.       Gun ia Pigh inoculation from specimen of renal tuberculosis endomaterium tuberculosis – Anim al killed after 6 weeks and examine for  TB.

4.       Allergic skin test (montoux test)MT.

5.       Histopathological examination  of biopsy from gland or endomaterium.

6.       Examination of CSF in case of meningitis (proten raised, sugar diminished, chloride normal >100 lymphocytes/cemm).

Serological test :- ELISA

Methods of preparation of stool for microscopic examination

 

 Methods of preparation of stool for microscopic examination

        Ans: The method for the preparation of stool for microscopy include:-

 (a). saline preparation: Take a little foecal material on a slide, Mix with normal saline (0.85%) tomake a thin emulsion. Cover it with a cover slip. The thickness should be such that one  should be able to see print through it.

(b). Iodine preparation: This is done as above using Gram’s iodine instead of saline. Iodine is used to examin the nuclear structure of cyst. It stain the chromatin granules of karysone of amoebic cyst brown. The glycogen vavoule also is stained brown. Iodine kills living material, motility of parasites will not be detected. In routine use iodine preparation is complementary to the saline preparation.

 (c). stool concentration method: These method are used to identify ova & cysts which are few in number and not detected by routine method.

                The method used are:-

(i). Flootation method: The stool is mixed with zinc sulfate or magnesium sulfate which has a  high sp gravity so that the parasite flouts in the solution. It is useful to                                           concentrate cysts, larval, most helminthic eggs.

                Zinc sulphate concentration method:-

                Reagents:- Zinc sulphate solution of sp gravity 1.80 is needed. It is prepared by                                dissolving 33gm of the chemical in 100ml of D/W.

                Method:- mix a small piece of stool with about 100ml of water or saline in a bottle or                        tube.

ü  Sieve the suspension into a beaker, through a strainer with small holes.

ü  Pour the contents of the beaker into a centrifuge tube.

ü  Centrifuge at 2000-3000 rpm/min for 1 min.

ü  Pour off the supernatant fluid.

ü  Resuspend the deposit in clean water and add enough water to fill the tube.

ü  Mix will and re-centrifuge.

ü  Pour of the supernatant fluid.

ü  Resuspunded in zinc sulphate solution, fill the test tube with solution.

ü  Centrifuge at high speed for min.

ü  Transfer the contents from the surface of the tube to a slide using a bacteriological wire loop. This surface film must be removed immediately.

ü  Add small drops of saline and mix.

ü  Cover with a coverslip.

ü  Examine under 10x and 40x objective.

Sterilization

 

 Sterilization

Sterilization means freeing of any object or subject from all living organism either in vegetative  or spore that is known as to be sterilized.

Sterilization can be classified as:

        (i). Physical method

        (ii). Chemical method

v  Physical method of sterilization: Under physical method the physical agents acts either by giving energy in the form of heat or radiation or by removing organism through filteration various physical agent are:-

 (a). Heat: Heat energy can be applied in two ways.

                (i). Dry heat

                (ii). Moist heat

(i). Dry heat: It kills the micro organism by causing destructive oxidation of essential cell       constituents or by protein denaturation. Dry heat is done by:-

ü  Flaming: The articles are passed through the Bunsen flame for few minutes like  sterilization of scalpels, mouth of culture tubes, glass slide etc.

ü  Red heat: It is done by holding the article directly into flame of burner until it becomes red hot like sterilization of point of forceps, spatula, inoculation wire etc.

ü  Hot air oven: This is most common method of sterilization by dry heat. In this process the bacteria are killed by oxidation of cell constituency like sterilization of glassware like tubes, glass, bottles, flasks, petridishes or powders etc.

(ii). Moist heat: It kills micro-organism by denaturation and coagulation of protein and enzymes. It can be done by various method.

ü  Pasteurization: Sterilization of milk is called pasteurization the milk is pasteurized either at 63⁰ c-66⁰c for 30 minutes (holding method) and other method is flash method which is done by heating at 72⁰c for 24 sec. followed by rapid cooling to 13⁰c or low. It is called high temp short time method.

Eg:- M-boris, Salmonella, Streptococcus, Brucella.

ü  Boiling: The material to be sterilized is kept in a contains and boil for 10 minutes. It kills all form of vegetative bacteria.

ü  Tyndallization: An exposure to 100⁰c for 20 minutes followed by intermittent heating for three successive days is known as tydallization. It is generally used to kill the spore forming bacteria. First day kills vegetative form of bacteria. Second day kill spore vegelative third complete kill.

ü  Autoclaving: It is moist frequently used method of sterilization. This is very reliable  method and sterilize the suitable culture media bacteriotogical media having no egg or serum, rubber bag, dressing, instuments etc.

(b). Radiation:-

                They are of two types:-

                (i). Non-ionising Radiation

                (ii). Ionising Radiation

 (i). Non- Ionising Radiation: Infrared radiation act as a hot air sterilization. It is used in the sterilization of syrinjes and catheters before packing or radiation is operation theaters and laboratories.

 (ii). Ionising Radiation: It kill the micro organism by attacking on their DNA and breaking  covalents bonds in DNA-X-ray kills vegetative cell readily. It is used in medicines for sterilization of heat sensitive items, such as sutures and surgical gloves, plastic syringes, swab, catheters etc.

  (c). Filteration: Filteration is most officient method used for the sterilization of heat labile fluids such as serum, antibiotic solution and some culture media containing serum and egg etc. This method is not effective against viruses most commonly used filter is composed of nitrocellulose and has a pre size of 0.22um. The size retains all bacteria & spores. Some filter are Asbostos filter, sintered glass filter, membrane filter, chamber land filter, Earthen- ware filter.