Lesson 10: CULTURE METHODS Flashcards
A population of bacteria grown in the laboratory is referred to as a
Culture
used to obtain a pure culture of an infectious agent, and also for studies leading to the identification of the pathogen.
Subculturing
Contains only single type of bacteria
Pure Culture
Contains two or more different bacteria
Mixed Culture
Periodic transfer of bacterial culture to new media to keep the bacterial population growing
Subculturing
using practices and procedures to prevent contamination from pathogens. It involves applying the strictest rules to minimize the risk of contamination
Aseptic technique
Indications for various culture methods:
- Isolate bacteria in pure culture and identify the same by performing various tests.
- Demonstrate biochemical, antigenic, and other phenotypic and genomic properties of the isolated colonies.
- Demonstrate susceptibility of the isolated bacteria to antibiotics, bacteriophages, bacteriocins, etc.
- Prepare antigens for various uses.
- Maintain stock culture.
- Estimate viable counts.
The piles of bacterial cells observed after an incubation period are called
Colonies
Complex media like EMB and Mc Conkey are use to detect what kind of bacteria
Gram Negative bacteria only
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.
Streak Plate Method
Procedure in Streak Plate Method
- a loopful of the inoculum is placed near the peripheral area of the plate.
- The inoculum is then spread with the loop to about one-fourth of the plate with close parallel strokes.
- From the primary inoculum, it is spread thinly over the plate by streaking with the loop in parallel lines.
- 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.
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.
Streak Plate Method
Also called as carpet culture
Lawn Culture
Lawn or carpet culture method is used for
a. Antibiotic susceptibility testing by disk diffusion method
b. Bacteriophage typing
c. For preparation of bacterial antigens and vaccines
Liquid culture method is use for
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.
After incubation, provides a uniform growth of the bacterium.
Lawn culture
Procedure of lawn culture method
- 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.
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.
Stroke Culture
Antibiotic sensitivity testing
Lawn culture
The use of stab culture method
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
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.
Stab culture method
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.
Pour plate culture methods
Pour plate culture method procedure
- Carried out in tubes, each containing 15 mL of molten agar.
- The molten agar in tubes is left to cool in a water bath at 45°C.
- The inoculum to be tested is diluted in serial dilution.
- Then 1 mL each of
diluted inoculum is added to each tube of molten agar and mixed well. - The contents of tubes are poured into sterile Petri dishes and allowed to set.
- 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
In tubes, bottles or flasks may be inoculated by touching with a charged loop or by adding the inoculum with pipettes or syringes.
Liquid Culture
Shake culture method procedure
- 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.
- Withdraw the pipette or wire and flame the mouth of the tube if it has a cotton-wool plug.
- Replace the cap or plug and discard the pipette into disinfectant or flame the wire.
- 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.
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.
Aerobic culture method
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.
Shake culture method
require incubation without oxygen and differ in their
requirement and sensitivity to oxygen.
Anaerobic culture method
will not grow from small
inocula unless oxygen is absent and the Eh of the medium is low.
Obligate anaerobes
Different culture methods
- Streak plate
- Lawn culture
- Stroke culture
- Stab culture
- Pour plate culture
- Shake culture
- Liquid culture
Aerobic bacteria that is incubated in a normal incubator with added Carbon Dioxide
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.
Most studies and tests of the physiological, serological and other characters of bacteria are valid only when made on a
Pure Culture
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.
Surface plating
Methods of isolating pure culture
- Surface plating
- Use of selective, enrichment or indicator media
- 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 - use of selective growth condition such as:
a. Separation of bacteria with different temperature optima
b. Cultivation under aerobic or anaerobic conditions - Separation of motile from non-motile bacteria can be effected using Cragie’s tube
- Animal inoculation
- Filters
widely used for the isolation of pathogens from specimens such as feces, with varied flora.
Use of selective, enrichment, or indicator media
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.
Selective media
media such as selenite broth for Salmonella sp, favor the multiplication of particular species as a step towards their isolation in pure culture.
Enrichment media
selective media for diphtheria
bacillus
tellurite media
Enrichment media for Salmonella sp
selenite broth
media such as Willis and Hobbs medium for Clostridium sp, contain ingredients that change in appearance with particular organisms and so assist their isolation.
Indicator media
Indicator media for for
Clostridium sp
Willis and Hobbs medium
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.
Separation of bacteria with different temperature optima
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
Heating at 65°C for 30 minutes or at higher temperatures for shorter periods:
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.
Pretreatment of specimens with appropriate bactericidal substances
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.
Cultivation under aerobic or anaerobic conditions
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.
Separation of motile from non-motile bacteria can be effected using Cragie’s tube
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.
U-tube
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.
a mixture of organisms containing the pneumococcus, e.g. in sputum
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
Animal inoculation
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.
Anthrax bacilli
mucus that is coughed up
Sputum
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.
The tubercle bacillus
Bacteria of differing sizes may be separated by the use of this. These are widely used for separating viruses from bacteria.
Filters
