This document discusses the cultivation of bacteria in the laboratory. It describes the process of culturing bacteria, which involves allowing microbes to grow on artificial media to identify and observe them. Various types of culture media are discussed, including liquid, solid that can be liquefied, and solid that cannot be liquefied. Agar is commonly used as it melts at boiling temperature and solidifies when cooled, without affecting bacterial growth. General considerations for culturing bacteria, such as temperature, moisture, oxygen, pH, nutrients, and sterile conditions are also outlined.

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)