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Cultivation of bacteria

Cultivation of bacteria

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Cultivation of bacteria

  1. 1. CULTIVATION OF BACTERIA
  2. 2. • CULTURING---PROCESS OF ALLOWING MICROBES TO GROW ONARTICIAL MEDIUM/MEDIA IN ORDER TO IDENTIFY THEM AND TO OBSERVE THEIR GROWTH. • CULTURE---REFERS TO THE GROWTH OF MICROBES • CULTURE MEDIUM---NUTRIENT SUBSTANCE WHERE MICROBES ARE GROWN
  3. 3. • GENERAL CONSIDERATIONS WHEN CULTURING ON ARTIFICIAL LAB. MEDIA: • 1. PROPER TEMPERATURE • 2. RIGHT AMOUNT OF MOISTURE • 3. REQUIRED OXYGEN TENSION • 4. PROPER pH • 5. NUTRIENTS & GROWTH-PROMOTING FACTORS
  4. 4. • 6. STERILECONDITION/FREE FROM CONTAMINANTS • BASIC TYPES OF CULTURE MEDIA: • 1. LIQUID • 2. SOLID THAT CAN BE LIQUIFIED BY HEATING • 3. SOLID THAT CANNOT BE LIQUIFIED
  5. 5. AGAR---COMMONLY USED CULTURE MEDIUM • USES OF AGAR AS A MEDIUM: • 1. MELTS AT BOILING TEMPERATURE • 2. SOLIDIFIES WHEN COOLED DOWN TO 40 DEGREES CELCIUS • 3. NO EFFECT ON BACTERIAL GROWTH • 4. IS NOT ATTACKED BY THE BACTERIA GROWING ON IT
  6. 6. ENRICHING MATERIALS • 1. CARBOHYDRATES (CHO)---ADDED TO INCREASE THE NUTRITIVE VALUE OF THE MEDIUM • 2. SERUM---USED TO INDICATE THE GROWTH OF LESS HARDY ORGANISMS • EX. WHOLE BLOOD/ ASCITIC FLUID • 3. DYES---USED AS INDICATORS TO DETECTACID FORMATION; INHIBITORS OF
  7. 7. • CERTAIN BACTERIA • EX. PHENOL RED---INDICATOR DYE • GENTIAN VIOLET---INHIBITORY DYE ( INHIBITS THE GROWTH OF MOST GRAM POSITIVE BACTERIA)
  8. 8. CLASSIFICATION OF CULTURE MEDIA • 1. DIFFERENTIAL---WHEN THE INGREDIENTS OF THE MEDIUM IS USED TO DISTINGUISH ORGANISMS GROWING TOGETHER. • EX. A. EMB (EOSIN METHYLENE BLUE) AGAR • B. MAC CONKEY AGAR---USED IN THE DIFFERENTIATION OF GRAM (-) BACTERIA OF THE INTESTINAL TRACT • C. LACTOSE---SEPARATES ORGANISMS THAT
  9. 9. • FEREMENT THE LACTOSE SUGAR FROM THOSE WHO DO NOT USE THE SUGAR • A. LACTOSE FERMENTERS---PRODUCES DEEPLY COLORED RED COLONIES WITH A METALLIC SHEEN • B. NON-FERMENTERS----PRODUCES COLORLESS COLONIES
  10. 10. • 2. SELECTIVE MEDIUM---PROMOTES THE GROWTH OF 1 ORGANISM & RETARD THE GROWTH OF OTHERS • A. DEOXYCHOLATE-CITRATE AGAR---INHIBITS THE GROWTH OF MOST COLIFORMS INCLUDING MANY STRAINS OF PROTEUS BUT FAVORS THE ISOLATION OF INTESTINAL PATHOGENS LIKE SALMONELLA & SHIGELLA
  11. 11. • B. LOWENSTEIN-JENSEN MEDIUM--- PROMOTES THE GROWTH OF TUBERCLE BACILLI BUT RETARDS THE GROWTH OF OTHER ORGANISMS • C. EMB AGAR/ MAC CONKEY AGAR---DISPLAY SELECTIVE FUNCTION IN THAT THEY ALLOW THE GROWTH OF GRAM-NEGATIVE BACTERIA BUT PREVENTS THE GROWTH OF GRAM- POSITIVE ONES
  12. 12. • SELECTIVE & DIFFERENTIAL MEDIA ---ARE OF • GREAT VALUE IN THE DIAGNOSIS OF INFECTIONS AS VARIOUS PATHOGENS TEND TO BE MIXED WITH MANY OTHER ORGANISMS.
  13. 13. CULTURE METHODS • A. PREPARATION OF CULTURE MEDIUM: • 1. DETERMINE ORGANISM TO BE CULTURED AND USE THE REQUIRED /SPECIFIC MEDIUM • 2. STERILIZATION OF ALL INSTRUMENTS NEEDED INCLUDING THE CULTURE MEDIUM BY AUTOCLAVING----MOST COMMONLY USED STERILIZATION PROCEDURE IN THE LAB.
  14. 14. • * **121-123 DEGREES CELCIUS TEMPERATURE • ***15-17 PSI PRESSURE • ***NOT USED IN THE STERILIZATION OF MATERIALS THAT ARE DESTROYED BY TOO MUCH HEAT. • B. INOCULATION---INTRODUCTION OF THE SOURCE OF THE ORGANISM
  15. 15. SOURCES OF INNOCULUM • 1. SPUTUM • 2. URINE • 3. BLOOD • 4. PUS • 5. RESPIRATORY OR UROGENITAL SECRETIONS • *** THESE INNOCULUM MAY BE ADDED TO A FLUID MEDIUM OR RUBBED GENTLY OVER THE SURFACE OF A SOLID MEDIUM USING A COTTON SWAB OR A WIRE LOOP
  16. 16. • C. ROUTINE INCUBATION PERIOD IS WITHIN 24 TO 48 HOURS WITH A TEMPERATURE OF ABOUT 0.5 EGREES CELCIUS & KEPT CONSTANT FROM DAY TO DAY • ***INSPECTION / STUDY OF CULTURES: • 1. LIQUID MEDIA LIKE NUTRIENT BROTH---TO OBSERVE FOR TURBIDITY, GAS PRODUCTION & COLOR CHANGE; PROCEED TO GRAM STAINING
  17. 17. • 2. SOLID MEDIUM----BACTERIA TEND TO CLING TOGETHER TO FORM A COLONY WHICH HAS CHARACTERISTICS LIKE TEXTURE, SIZE, SHAPE, COLOR, ELEVATION, ETC THAT ARE FAIRLY CONSTANT FOR EACH SPECIE • D. DISCARDING CULTURES--VIA AUTOCLAVING OR INCINERATION
  18. 18. CULTIVATION OF BACTERIA IN THE LABORATORY • 1. ALL INSTRUMENTS/EQUIPMENTS ARE STERILIZED FOR THE STUDENTS • 2. FOR SOLID MEDIUM, USE EITHER AGAR PLATE OR AGAR SLANT • 3. OBTAIN INOCULUM FROM EITHER THE COIN OF ANY DENOMINATION, FROM THE TABLE TOP[,BALLPEN, HEM OF PANTS OR SKIRT, IN BETWEEN TOES, ARMPITS & FRUIT PEELINGS
  19. 19. • 4. DO SERIAL DILUTION----MAKES BACTERIAL PLATE COUNT MORE ACCURATE WITH THE PREMISE THAT EVERY BACTERIUM PRESENT IN AN INOCULUM DEVELOPS INTO A COLONY • 5. PROCEED TO POUR PLATING OR STREAK PLATING TECHNIQUES • 6. BACTERIAL PLATE COUNT USING THE QUEBEC COLONY COUNTER
  20. 20. • QUANTIFICATION----HELPS TO INDICATE WHETHER BACTERIA RECOVERED FROM A SAMPLE (URINE) ARE PATHOGENIC OR JUST PLAIN CONTAMINANTS ( LESS THAN 10,000 COLONIES PER ML REPRESENT NORMAL FLORA OR JUST CONTAMINANTS)

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