Lesson 10: CULTURE METHODS

Question 1

A population of bacteria grown in the laboratory is referred to as a

Answer 1

Culture

Question 2

used to obtain a pure culture of an infectious agent, and also for studies leading to the identification of the pathogen.

Answer 2

Subculturing

Question 2

Contains only single type of bacteria

Answer 2

Pure Culture

Question 3

Contains two or more different bacteria

Answer 3

Mixed Culture

Question 4

Periodic transfer of bacterial culture to new media to keep the bacterial population growing

Answer 4

Subculturing

Question 5

using practices and procedures to prevent contamination from pathogens. It involves applying the strictest rules to minimize the risk of contamination

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Aseptic technique

Question 6

Indications for various culture methods:

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1. Isolate bacteria in pure culture and identify the same by performing various tests.
2. Demonstrate biochemical, antigenic, and other phenotypic and genomic properties of the isolated colonies.
3. Demonstrate susceptibility of the isolated bacteria to antibiotics, bacteriophages, bacteriocins, etc.
4. Prepare antigens for various uses.
5. Maintain stock culture.
6. Estimate viable counts.

Question 6

The piles of bacterial cells observed after an incubation period are called

Answer 6

Colonies

Question 7

Complex media like EMB and Mc Conkey are use to detect what kind of bacteria

Answer 7

Gram Negative bacteria only

Question 7

The most effective way to isolate a single type of bacteria from a source that contains many by diluting the individual cells by spreading them over the surface of an agar plate using a platinum or inoculating loop of 2–4 mm diameter. On the plate single cells reproduce and create millions of clones, which all pile up on top of the original cell.

Answer 7

Streak Plate Method

Question 8

Procedure in Streak Plate Method

Answer 8

1. a loopful of the inoculum is placed near the peripheral area of the plate.
2. The inoculum is then spread with the loop to about one-fourth of the plate with close parallel strokes.
3. From the primary inoculum, it is spread thinly over the plate by streaking with the loop in parallel lines.
4. The inoculated culture plate is incubated at 37°C overnight
   for demonstration of colonies.

Note: The loop is flamed and cooled in between the streaks to obtain isolated colonies.

Question 9

Confluent growth occurs at the primary inoculum, but becomes progressively thinner, and well-separated colonies are demonstrated on the final streaks of the inoculum Single isolated colonies obtained by this method are very useful to study various properties of bacteria.

Answer 9

Streak Plate Method

Question 9

Also called as carpet culture

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Lawn Culture

Question 9

Lawn or carpet culture method is used for

Answer 9

a. Antibiotic susceptibility testing by disk diffusion method
b. Bacteriophage typing
c. For preparation of bacterial antigens and vaccines

Question 9

Liquid culture method is use for

Answer 9

a. blood culture and for sterility
b. dilution in the medium
c. large yields culture.

Note: liquid cultures does not provide a pure culture from mixed inocula—the major disadvantage, nor identify a bacteria.

Question 9

After incubation, provides a uniform growth of the bacterium.

Answer 9

Lawn culture

Question 9

Procedure of lawn culture method

Answer 9

• flood the surface of the plate with a liquid culture or suspension of the bacterium, pipetting off the excess inoculum and incubating the plate.
• Alternatively, the surface of the plate maybe inoculated by applying a swab soaked in the bacterial culture or suspension.

Question 9

provides a pure growth of bacteria for carrying out slide agglutination and other diagnostic tests. It is carried out in tubes usually containing slanted nutrient agar slopes.

Answer 9

Stroke Culture

Question 10

Antibiotic sensitivity testing

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Lawn culture

Question 10

The use of stab culture method

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a. demonstration of gelatin liquefaction
b. demonstration of oxygen requirement of the bacterium under study
c. for the maintenance of stock cultures
d. to study motility of bacteria in semisolid agar

Question 10

a suitable medium such as nutrient gelatin or glucose agar is punctured with a long, straight, charged wire into the center of the medium and withdrawing it in the same line to avoid splitting the medium. The medium is allowed to set, with the tube in the upright position, providing a flat surface at the top of the medium.

Answer 10

Stab culture method

Question 10

used to determine approximate number of viable organisms in liquids, such as water or urine. It is used to quantitate bacteria in urine cultures and also to estimate the viable bacterial count in a suspension.

Answer 10

Pour plate culture methods

Question 10

Pour plate culture method procedure

Answer 10

1. Carried out in tubes, each containing 15 mL of molten agar.
2. The molten agar in tubes is left to cool in a water bath at 45°C.
3. The inoculum to be tested is diluted in serial dilution.
4. Then 1 mL each of
   diluted inoculum is added to each tube of molten agar and mixed well.
5. The contents of tubes are poured into sterile Petri dishes and allowed to set.
6. After overnight incubation of these Petri dishes at 37°C, colonies are found to be distributed throughout the depth of the medium, which can be counted using a colony counter

Question 10

In tubes, bottles or flasks may be inoculated by touching with a charged loop or by adding the inoculum with pipettes or syringes.

Answer 10

Liquid Culture

Question 10

Shake culture method procedure

Answer 10

1. melt nutrient agar in a test tube, cooling it to 45°C and inoculating it while molten from a liquid medium with a drop from a capillary pipette or a wetted straight wire, depending on the desired size of the inoculum. 2. Withdraw the pipette or wire and flame the mouth of the tube if it has a cotton-wool plug. 3. Replace the cap or plug and discard the pipette into disinfectant or flame the wire. 4. Mix the contents of the tube by rotation between the palms of the hands before the agar solidifies. Incubate it at 37°C for 24 hours and look for the growth of the organisms.

Question 10

The incubation is done in an incubator under normal atmospheric condition at 37°C is standard practice in the culture of bacteria pathogenic to man. Brucella abortus and capnophilic streptococci require extra CO2 in the air in which they are grown and others, such as the pneumococcus and gonococcus, grow better in air supplemented with 5 to 10 percent CO2.

Answer 10

Aerobic culture method

Question 10

a deep culture of agar or gelatin through which the inoculum is evenly distributed by shaking before the medium is solidified and which is used chiefly for the demonstration of anaerobic colonies.

Answer 10

Shake culture method

Question 10

require incubation without oxygen and differ in their requirement and sensitivity to oxygen.

Answer 10

Anaerobic culture method

Question 10

will not grow from small inocula unless oxygen is absent and the Eh of the medium is low.

Answer 10

Obligate anaerobes

Question 10

Different culture methods

Answer 10

1. Streak plate 2. Lawn culture 3. Stroke culture 4. Stab culture 5. Pour plate culture 6. Shake culture 7. Liquid culture

Question 10

Aerobic bacteria that is incubated in a normal incubator with added Carbon Dioxide

Answer 10

Brucella abortus and capnophilic streptococci and others, such as the pneumococcus and gonococcus, that grow better in air supplemented with 5 to 10 percent CO2.

Question 11

Most studies and tests of the physiological, serological and other characters of bacteria are valid only when made on a

Answer 11

Pure Culture

Question 11

a method routinely employed in clinical bacteriology and enables the isolation of distinct colonies which may be picked out, if necessary for further purification and study.

Answer 11

Surface plating

Question 11

Methods of isolating pure culture

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1. Surface plating 2. Use of selective, enrichment or indicator media 3. Selective treatment of the specimen before culture such as: a. Heating at 65°C for 30 minutes or at higher temperatures for shorter periods and; b. Pretreatment of specimens with appropriate bactericidal substances 4. use of selective growth condition such as: a. Separation of bacteria with different temperature optima b. Cultivation under aerobic or anaerobic conditions 5. Separation of motile from non-motile bacteria can be effected using Cragie’s tube 6. Animal inoculation 7. Filters

Question 11

widely used for the isolation of pathogens from specimens such as feces, with varied flora.

Answer 11

Use of selective, enrichment, or indicator media

Question 12

media such as tellurite media for the diphtheria bacillus, have been devised so that, the majority of organisms likely to be associated with those for which the media are used will not grow, and the isolation of pure cultures is thus facilitated.

Answer 12

Selective media

Question 12

media such as selenite broth for Salmonella sp, favor the multiplication of particular species as a step towards their isolation in pure culture.

Answer 12

Enrichment media

Question 13

selective media for diphtheria bacillus

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tellurite media

Question 14

Enrichment media for Salmonella sp

Answer 14

selenite broth

Question 15

media such as Willis and Hobbs medium for Clostridium sp, contain ingredients that change in appearance with particular organisms and so assist their isolation.

Answer 15

Indicator media

Question 16

Indicator media for for Clostridium sp

Answer 16

Willis and Hobbs medium

Question 17

The temperature and atmosphere chosen for a culture automatically preclude the growth of many bacteria. Incubation at 37°C, used for most medically important bacteria, is too warm for some air contaminants, which subsequently appear as colonies when plates are kept at room temperature. Some pathogens are selectively favoured by growth at temperatures above 37ºC. Only thermophilic bacteria grow at 60ºC.

Answer 17

Separation of bacteria with different temperature optima

Question 18

can be used to separate spores from vegetative bacilli but does not guarantee that spores will germinate under subsequent cultural conditions. For example, by heating a mixture containing vegetative and spore forming bacteria at 80°C the former can be eliminated. This method is useful for the isolation of tetanus bacilli from dust and similar sources

Answer 18

Heating at 65°C for 30 minutes or at higher temperatures for shorter periods:

Question 19

destroy the unwanted bacteria. This method is the standard practice for the isolation of tubercle bacilli from sputum and other clinical specimens, by treatment with alkali, acid or other substances to which most commensals are susceptible but tubercle bacilli are resistant.

Answer 19

Pretreatment of specimens with appropriate bactericidal substances

Question 20

Obligate aerobes and anaerobes may be separated by cultivation under aerobic or anaerobic conditions. Strict anaerobes will not grow in air and most facultative anaerobes grow less vigorously under anaerobic than under aerobic conditions.

Answer 20

Cultivation under aerobic or anaerobic conditions

Question 21

This consists of a tube of semisolid agar, with a narrow tube open at both ends placed in the center of the medium in such a way that it projects above the level of the agar. Inoculation of the mixture is made into the central tube. On incubation, subculture is taken from the surface of the medium in the outer tube because the motile bacteria alone traverse the agar and appear at the top of the medium outside the central tube.

Answer 21

Separation of motile from non-motile bacteria can be effected using Cragie’s tube

Question 22

also serves the same purpose as the Cragie's tube, inoculation being performed in one limb and the subculture taken from the other. This method can also be used to obtain phase variants in Salmonella species.

Answer 22

U-tube

Question 23

inoculated subcutaneously into a mouse, the animal dies of pneumococcal septicemia in 12 to 48 hours and the organism can be obtained in pure culture from the heart blood.

Answer 23

a mixture of organisms containing the pneumococcus, e.g. in sputum

Question 23

Pathogenic bacteria may be isolated from mixtures by inoculation into appropriate animals due to the fact that laboratory animals are highly susceptible to certain organisms for example, the mouse to the pneumococcus

Answer 23

Animal inoculation

Question 24

can be distinguished from other aerobic sporulating bacilli by inoculation into mice or guinea pigs. Produce a fatal septicemia and may be cultured pure from the heart blood.

Answer 24

Anthrax bacilli

Question 24

mucus that is coughed up

Answer 24

Sputum

Question 25

can be isolated from contaminating organisms by inoculation of an infected specimen into a guinea pig. Found in a pure state in the resulting lesions.

Answer 25

The tubercle bacillus

Question 26

Bacteria of differing sizes may be separated by the use of this. These are widely used for separating viruses from bacteria.

Answer 26

Filters