1. PATHOLOGICAL TOOLS AND TECHNIQUES

2. Clearing & preparation of glassware  Glassware are boiled in hot water before washing.  Sock the glassware in soap water for 10 minutes and wash them with soft brush or cotton.  Clear glassware in tap water.  Transfer the glassware to the pot containing chromic acid solution for about 30 minutes or over night.  Wrap the paper around glassware and keep it in oven at 60 0 C temperature for over night.  clear the glassware with 70% alcohol before using.

3. Culture media The growth of an organisms on a medium called culture and the food base that support the growth of an organism is called culture media. Culture media are those used on the basis of experience and not on the basis of exact knowledge of their composition and action (e.g. milk, diluted blood, vegetable juices) called Natural Media. A number of culture media, were developed with exact knowledge of nutritional requirements of microorganisms. These are chemically defined media with exactly known composition is called Synthetic Media.

4. Natural medium  Potato Dextrose Agar media (PDA) Potato extract – 450 ml Dextrose – 9.0 gm Peptone – 1.0 gm Agar – 15.0 gm Distilled water – 475 ml  Corn Meal Dextrose Agar media Corn extract – 200 ml Dextrose – 10.0 gm Peptone – 20.0 gm Agar – 20.0 gm Distilled water – 800 ml

5. Synthetic medium  Asthana and Hawkers media Glucose – 5.0 gm KNO 3 – 3.5 gm KH 2 PO 4 – 1.0 gm MgSO 4, 7H 2 O - 0.75 gm Agar – 20.0 gm Distilled water – 1000 ml  Czapeks Dox media NaNO 3 – 2.0 gm KH 2 PO 4 – 1.0 gm MgSO 4, 7H 2 O - 0.5 gm KCL - 0.5 gm Sucrose – 30.0 gm Agar – 20.0 gm Distilled water – 1000 ml

6. Sterilization Dry heat This is achieved in electric oven at 180 0 C for 2 hrs. Petri plate, Pipette, Needle and Metallic equipments are sterilized in this way.

7. Sterilization  Wet Air or Steam Culture media and water are sterilized by using moist heat. i.e. steam under pressure. It is done through autoclaving and also by pressure cooker.

8. Sterilization  Dried Flame By keeping on direct flame up to red hot and cooling in 10% alcohol. Repeat it 2-3 times. Inoculating needle, forceps, slides and glass rods are sterilized.

9. Sterilization  Surface disinfection Phenol, Boric acid, 0.1% solution of HgCl 2 is used to sterilized the surface of diseased tissues.  UV treatment UV rays are used to kill the microorganisms and keep the sterile inoculation chamber.  Fumigation Fumigation is used to make sterile atmosphere where UV can not be used or not available.

10. Pouring  Preparation for pouring  take Petri plate and clean them with absolute alcohol.  prepare the bundles of 5 Petri plates wrapping in newspaper.  sterilized the bundles in hot air oven at 180 0 C for 02 hours.  Melt autoclave media by heating on the water bath.

11. Pouring  Methods of pouring  wash the hand with soap water as well as absolute alcohol and get them air dry.  Arranged sterile Petri plates near the burner.  Hold the conical flask containing autoclave medium in right hand. Remove cotton plug through left hand near the burner / lamp.  Gently lift the lid of upper Petri plate with the left hand and pour about 10-20 ml medium.  Allow the culture media for solidification.

12. Inoculation of fungal culture  Arrange sterile Petri plates near the burner / lamp.  select the infected host tissue and cut into small pieces with the help of sterile blade.  Wash the pieces with 01% HgCl 2 solution and distilled water.  Place 2-4 sterile piece in a single previously poured Petri plate.  Incubate at room temperature i.e. 27 o C for growth in inverted position.  In case of soil suspension with sterile water in test tube and make serial dilution. For inoculation 5 th dilution of suspension poured into Petri plate.  In the air exposed poured plate add some tap water and incubate similarly.

13. Slants preparation  Pure fungal cultures are maintained in slants.  Slants are prepared by pouring 05-10 ml medium in sterile culture test tube and plugged with cotton plug.  Culture test tube are allowed to get solidify in slanting condition on wooden block.  This provide more surface for growth of an organism.  After 10 day of pure culture organism store in refrigerators.

14. Laboratory Precautions  Broken or chipped glassware should not be left on the bench or shelves or in the floor.  Chemicals bottles and apparatus should be kept in proper, after use.  Always wear cotton clothes in the lab.  Neutralize the acid spills with sodium bicarbonate and alkali spills with boric acid.  A person working in a chemical laboratory should locate fire extinguishers.  First aid box should be used for any injury.  Switch off the instruments after use.  One should perform only of the allotted experiments & should not work alone in the laboratory.  Special care should be taken while working with organic solvents and inflammable substances.  Use rubber gloves while working with toxic substances.  In case of fumes, use exhaust fans & avoid breathing in fumes.  Any dangerous laboratory situation should be brought immediately to the notice of the laboratory supervisor.  Clean your seat before leaving the laboratory.