Cultivation of bacteria

1. CULTIVATION OF BACTERIA

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. • 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. • 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. 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. 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. • CERTAIN BACTERIA
• EX. PHENOL RED---INDICATOR DYE
• GENTIAN VIOLET---INHIBITORY DYE ( INHIBITS
THE GROWTH OF MOST GRAM POSITIVE
BACTERIA)

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. • 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. • 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. • 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. • SELECTIVE & DIFFERENTIAL MEDIA ---ARE OF
• GREAT VALUE IN THE DIAGNOSIS OF
INFECTIONS AS VARIOUS PATHOGENS TEND
TO BE MIXED WITH MANY OTHER
ORGANISMS.

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. • * **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. 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. • 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. • 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. 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. • 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. • 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)