Controlling Microbial Growth in Vitro

Factors that affect microbial growth 1. Availability of nutrients- living organisms require nutrients. Appropriate nutrients must be available to survive in particular environment 2. Moisture- all living organisms require water to carry out their normal metabolic processes and most will die in environments containing too little moisture. 3. Temperature every microorganisms growth temperature Thermophiles - organisms that love heat. (minimum growth temperature: 25oC, optimum growth temperature: 50-60oC, maximum growth temperature: 113oC) Mesophiles - microbes that grow best at moderate temperatures (minimum growth temperature: 10oC, optimum growth temperature: 20-40oC, maximum growth temperature: 45oC) Psychrophiles - prefer cold temperatures. They thrive in cold ocean water.(minimum growth temperature:-5oC, optimum growth temperature: 10-20oC, maximum growth temperature: 30oC) Psychroduric - microbes that prefer warmer temperatures, but can tolerate or endure very cold temperatures and can be preserved in the frozen state. 4. pH- refers to the acidity and alkalinity of a solution. Most microbes prefer a neutral or slightly alkaline growth medium (pH 7.0 to 7.4) acidophiles- such as those that can live in highly acidic environments such as pH 2 to 5) alkaliphiles- prefer an alkaline environment 9pH greater than 8.5), such as is found inside the intestine(pH 9) 5. Gaseous atmosphere- to grow a particular microorganism in the laboratory, it is necessary to provide the atmosphere that it requires. Encouraging the growth of microorganisms in vitro (events outside the body) Culture- the growth of organisms obtained in a culture medium after its incubation period Culture medium- any material where microorganisms may thrive for their nourishment and reproduction Incubation period- is the time needed to let previously inoculated culture media to show a distinct colony or colonies within a desired temperature. It is also the time needed for the microorganisms to adapt, grow and multiply in a new environment. Colony - a group of microorganisms growing together characteristically in a culture medium Inoculum- the fished colony hanging on a wire loop, cotton swabs that is ready for transfer to another culture medium for cultivation. Types of culture: o Contaminated culture- a culture that accidentally contains one or more group of microorganisms which should not be there at all. o Mixed culture- there are two or more desired species of microorganism living in the culture medium o Pure culture- a culture that contains only one group of microorganisms.

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Culturing bacteria in the laboratory Bacterial growth refers to an increase in the number of organisms rather than an increase in their size. When each bacterial cell reaches its optimum size, it divides by binary fission into two daughter cells. On solid medium, binary fission continues through many generations until a colony is produced. A bacterial colony is a mound or pile of bacteria containing millions of cells. Binary fission continues for as long as the nutrient supply, water, and space allow and ends when the nutrients are depleted or the concentration of cellular waste products reaches a toxic level. Colony development on agar surfaces helps in the identification of the cultured microorganism. Typically, depending on the medium used, individual species of microorganisms form colonies of characteristic size and shape. Even when mixed population has been properly cultured, one can distinguish one from the other based on overall appearance of the colonies.

Culture Media The media that are used in microbiology laboratories to culture bacteria are referred to as artificial media or synthetic media, because they do not occur naturally; rather, they are prepared in the laboratory.

They are number of ways of categorizing the media that are used to culture bacteria:
Chemically defined medium- is one in which all the ingredients are known; this is because the medium was prepared in the laboratory by adding a certain number of grams of each of the components(carbohydrates, amino acids, salts) Complex medium- is one in which the exact contents are not known. Complex media contain ground up or digested extracts from animal organs (hearts, liver, and brains), fish, yeasts, and plants, which provide the necessary nutrients, vitamins and minerals. Liquid media- also known as broths are contained in tubes Ex.: thioglycollate broth is very popular liquid medium for use in the laboratory. THIO supports the growth of all categories of bacteria from obligate aerobes to obligate anaerobes.

Solid media - are prepared by adding agar to liquid media and then pouring the media into tubes or Petri dishes, where the media solidifies. Bacteria are then grown on the surface of the agar- containing solid media. A gar is a complex polysaccharide that is obtained from a red marine alga; it is used as a solidifying agent, much like gelatin is used as a solidifying agent in the kitchen. Enriched medium is a broth or slid medium containing a rich supply of special nutrients that promotes the growth of fastidious organisms (complex nutritional requirements) It is usually prepared by adding extra nutrients to a medium called nutrient agar.
Ex. Blood agar (nutrient agar plus 5% sheep red blood cells) and chocolate agar (nutrient agar plus powdered hemoglobin) Chocolate agar is used to culture important, fastidious, bacterial pathogens, like Neisseriae gonorrhoeae and Haemophilus influenzae.

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Selective medium has added inhibitors that discourage the growth of certain organisms without inhibiting growth of an organism being sought.
MacConkey agar inhibits the growth of gram positive bacteria and thus is selective for gramnegative bacteria. Phenylethyl alcohol agar (PEA) and colistin-nalidixic acid (CAN) agar inhibit the growth of Gramnegative bacteria and thus are selective for gram-positive bacteria Thayer Martin agar and Martin Lewis agar (chocolate agars containing extra nutrients plus several antimicrobial agents) are selective for Neisseria gonorrhoeae. Only salt tolerant (haloduric) bacteria can grow on mannitol salt agar (MSA)

Differential medium- permits the differentiation of organisms that grow on the medium.
MacConkey agar is frequently used to differentiate among various gram-negative bacilli that are isolated from fecal specimens. Gram-negative bacteria able to ferment lactose (an ingredient of MacConkey agar) produce pink colonies, whereas those unable to ferment lactose produce colorless colonies. It differentiates between lactose-fermenting and non-lactose fermenting gram negative bacteria. Mannitol salt agar is used to screen for Staphylococcus aureus; not only will S. aureus grow on MSA, but it turns the originally pink medium to yellow because of its ability to ferment mannitol. Blood agar is also a differential medium because it is used to determine the type of hemolysis that bacterial isolate produces.

Inoculation of Culture Media

Inoculation of a liquid medium involves adding a portion of the specimen to the medium. Inoculation of a solid or plated medium involves the use of a sterile inoculating loop to apply a portion of the specimen to the surface of the medium, process called streaking.

Importance of using Sterile Technique:

sterile technique is practiced when it is necessary to exclude all microorganisms from a particular area, so that the area will be sterile. Such unwanted microbes are referred to as contaminants. Minimizes the possibility of contamination and protects the laboratory worker from becoming infected.

Incubation
After media are inoculated, they must be incubated. Incubator contains the appropriate atmosphere and moisture level and is set to maintain the appropriate temperature. To culture most human pathogens, the incubation is set at 35 to 37oC.

3 TYPES OF INCUBATOR 1. A carbon dioxide incubator concentration of 5 to 10% carbon dioxide. To isolate capnophiles. 2. A non CO2 incubator is an incubator containing room air; thus it contains atmosphere devoid of oxygen. 3. an anaerobic incubator is an incubator containing an atmosphere devoid of oxygen.

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Bacterial Population growth curve A population growth curve for any particular species of bacteria may be determined by growing a pure culture of the organism in a liquid medium at a constant temperature. The growth curve four phases: 1. Lag phase- bacteria absorb nutrients, synthesize enzymes and prepare for cell division. The bacteria do not increase in number during the lag phase 2. Logarithmic growth phase- the bacteria multiply rapidly that the umber of organisms doubles with each generation time. Growth rate is the greatest during the log phase. The log phase is always brief, unless the rapidly dividing culture is maintained by constant addition of nutrients and frequent removal of waste products 3. stationary phase -as the nutrients in the liquid medium are used up and the concentration of toxic waste products from the metabolizing bacteria build up, the rate of division slows, such that the number of bacteria that are dividing equals the number that are dying. 4. Death phase - as overcrowding occurs the concentration of toxic waste products continues to increase and the nutrient supply decreases. The microorganisms die at a rapid rate.

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