Culture and Culture Media PDF

Culture and Culture Media MS. REGINA MARIE TUQUIB CULTURES ARE THE GROWTH OF MICROORGANISMS IN A CULTURE MEDIUM. SINCE THE STUDY OF MICROORGANISMS RELIES ON THEIR ABILITY TO GROW AND SURVIVE OUTSIDE THE HOST, THE USE OF CULTURE METHODS IS CERTAINLY EFFICIENT. UTILIZING EFFECTIVE AN...

Culture and Culture Media MS. REGINA MARIE TUQUIB CULTURES ARE THE GROWTH OF MICROORGANISMS IN A CULTURE MEDIUM. SINCE THE STUDY OF MICROORGANISMS RELIES ON THEIR ABILITY TO GROW AND SURVIVE OUTSIDE THE HOST, THE USE OF CULTURE METHODS IS CERTAINLY EFFICIENT. UTILIZING EFFECTIVE AND APPROPRIATE CULTURE MEDIUM FOR GROWTH, TRANSPORT, AND STORAGE FACILITATES THE STUDY. A culture medium is composed of a mixture of nutrients such as carbon, nitrogen, sulfur, phosphorus, hydrogen, oxygen, and buffers. A liquid, semi-solid, or solid medium can be utilized to observe the growth patterns of microorganisms as well as their transport and storage. Inhibitory agents like dyes, salts, and antimicrobials are added into the medium to facilitate the isolation of the desired organism while suppressing the growth of other organisms present in the sample.  THE SELECTION OF THE MEDIUM FOR INOCULATION IS BASED ON THE TYPE OF SPECIMEN SUBMITTED FOR CULTURE AND THE KIND OF ORGANISMS THAT ARE INVOLVED IN THE INFECTION PROCESS.  THE SELECTION OF PRIMARY CULTURE MEDIA IS SOMEWHAT STANDARDIZED FOR THE ROUTINE BACTERIAL CULTURE.  IF SEVERAL PLATES WILL BE INOCULATED FOR A GIVEN SPECIMEN, THE MEDIA SHOULD BE ARRANGED STARTING WITH THE MOST ENRICHED TO THE MOST SELECTIVE ONE. IN THIS MANNER, INHIBITORY SUBSTANCES ADDED TO THE SELECTIVE MEDIA WILL NOT BE CARRIED OVER FROM ONE MEDIUM TO ANOTHER. ACCORDING TO COMPOSITION 1. Synthetic or defined medium  It is a medium in which all the components are known.  It is used for research purposes as either a liquid or solid medium.  It is preferred for the isolation of cyanobacteria and chemoorganotrophs.  An example is the BG-11 medium. ACCORDING TO COMPOSITION 2. Non-synthetic or complex medium  It is a medium in which some of the substances are unknown (peptones, meat, and yeast extracts).  It is very useful for the isolation of medically significant bacteria.  Some examples are nutrient broth (NB) medium, TSB, and MAC agar.  ACCORDING TO COMPOSITION 3. Tissue culture medium  It used for obligate intracellular bacteria (Rickettsia and Chlamydia).  Some examples are W138 cells, 229 cells, and McCoy cells.  HeLa 229 cells are human cervical tissue while McCoy cells and W 138 cells are fibroblasts. All of these are used for the isolation of Chlamydia.  Embryonated eggs are utilized for the propagation of Rickettsia.  ACCORDING TO THE DISPENSING OR DISTRIBUTION METHOD FOR THE MEDIUM  Plated media are distributed into the dish or plate.  Tubemedia are prepared as either liquid, slant, butt and slant, or butt.  Examples are triple sugar iron (TSI) agar, SIM, Simmons’ citrate agar (SCA) and lysine iron sugar (LIA).  ACCORDING TO USE 1. Simple media, general purpose media, and supportive media  These are routinely used in the laboratory and without additional supplements.  Thesemedia are usually composed of meat and soybean extracts.  Some examples are nutrient agar (NA), nutrient broth (NB), and TSB.  ACCORDING TO USE 2. Enrichment media (Liquid-type  These can also be used as media) supplement to agar plates  These are used to propagate the to detect aerobes, growth of a certain group of anaerobes, and bacteria from a mixture of microaerophiles organisms. (thioglycollate).  These contain specific nutrients and  Some examples are without additional supplements. alkaline peptone water,  These media are incubated for a selenite F, thioglycollate, certain period and then tetrathionate, Gram- subcultured to isolate the desired negative (GN) broth, and organism. Lim broth.  ACCORDING TO USE 3. Enriched media and non-selective media  These are media with additional supplements such as blood, vitamins , and yeast extract, which are necessary for the growth of fastidious organisms.  These are solid-type media.  Some example are BAP and CAP.  BAP is used to differentiate the hemolytic patterns of bacteria.  CAP is routinely used for the recovery of Haemophilus species.  BAP and CAP are used for the isolation of fastidious organisms.  ACCORDING TO USE 4. Differential Media  These media allow the visualization of metabolic differences between groups of bacteria.  Some examples are MAC, BAP, eosin methylene blue (EMB), and Hektoen enteric agar (HEA).  MAC differentiates lactose fermenters (pink colonies) from non-lactose fermenters (colorless colonies).  BAP differentiates hemolytic patterns of streptococci.  Neutral red is the pH indicator in MAC that detects the fermentation of sugar and eventually the production of acid.  ACCORDING TO USE 5. Selective Media  Thesemedia are incorporated with antibiotics, dyes, or chemicals to inhibit the growth of other organisms while promoting the growth of the desired organism.  Some examples are HEA, MAC, xylose lysine desoxycholate (XLD) agar, bismuth sulfite agar (BSA), mannitol salt agar (MSA), and Thayer-Martin agar (TMA).  ACCORDING TO USE Other Selective Media a. Gentamicin blood agar for Streptoccocus b. Bacitracin chocolate agar for Haemophilus c. Blood agar plate with ampicillin for Aeromonas d. Phenylethyl alcohol for Gram-positive bacteria e. Columbia colistin-nalidix acid (CNA) agar for Gram- positive bacteria  ACCORDING TO USE THERE ARE INHIBITORY SUBSTANCES THAT ARE ADDED TO THE CULTURE MEDIA TO ISOLATE THE DESIRED ORGANISM AND MAKE A MEDIUM SELECTIVE: a. For Gram-positive bacteria: Crystal/Gentian violet, basic/carbol fuchsin, and bile salt b. For Gram-negative bacteria - Potassium tellurite and sodium azide c. For swarming bacteria - Alcohol and chloral hydrate  ACCORDING TO USE 6. Special Media  It is used to isolate bacteria with specific growth requirements.  Some examples are Löwenstein-Jensen (LJ) medium and thiosulfate-citrate-bile salts-sucrose (TCBS) agar.  LJ medium is a protein-rich medium that is composed of whole eggs and malachite green and which supports the growth of mycobacteria; it is sterilized by inspissation and not by autoclaving.  TCBS is selective for the isolation of Vibrio in which sterilization is performed by boiling, and never by autoclaving, and thus considered as a "special medium."  INOCULATION OF SPECIMEN  Sterile body fluids, pus, urine, and sputum are inoculated directly into the selected media.  Specimens received on swabs can be inoculated directly into the culture media.  Specimens that require a direct or "bedside" inoculation are blood, genital specimens, corneal scrapings, sterile fluids like synovial and peritoneal fluids, and nasopharyngeal swabs for isolation of Bordetella pertussis.  INOCULATION TECHNIQUES  Streaking is the most common  The stabbing of the medium manner of inoculation. is usually performed with  A specimen that is collected group A streptococci to through a swab is inoculated by gently "rolling the tip of the create anaerobiosis and swab" onto the upper portion promote sub-surface of the plate; the inoculated hemolysis. area should be streaked by a sterile loop afterwards.  Overlapping inoculation is  The placement of fluid used for antimicrobial specimens or swabs into broth sensitivity test (disk diffusion or liquid media. method).  MANNER OF INOCULATION  Theinoculating loop is sterilized and allowed to cool thoroughly before use.  The inoculating loop should be flamed between different media plates, except when the specimen is collected with swabs.  Urine specimens are inoculated using a quantitative isolation technique with a calibrated loop (0.01 mL or 0.001 mL) to deliver a specified volume.  INCUBATION CONDITIONS  Aerobes - 21% O₂ + 0.03% CO₂  Anaerobes - 0% O₂, 5%-10% CO₂ + 5%—10% H₂ + 80%-90% N₂ (anaerobe jars, bags, or chambers)  Capnophiles - 5%-10% CO₂ + 15% O₂ (CO₂ incubator or bag; 3% CO₂ in candle jars)  Microaerophiles - 5%-10% O₂ + 8%-10% CO₂  ANAEROBIC CULTIVATION Ways to Facilitate Anaerobic Cultivation 1. The use of a special culture medium incorporated with thioglycollate and cysteine (reducing agents). 2. The boiling of culture medium to remove (drive off) oxygen. 3. The use of an anaerobic chamber system with a vacuum pump and nitrogen gas to remove residual oxygen. 4. The use of a gas-pak jar containing a palladium catalyst. 5. For small-volumes, plastic bags or pouches containing calcium carbonate and catalyst can be used.  ANAEROBIC CULTIVATION Components of an Anaerobic Chamber 1. Nitrogen gas - is used as a filler for the remaining percentage of the anaerobic atmosphere. 2. Palladium pellets - are used to remove residual oxygen from the chamber by combining with hydrogen to form water. 3. Silica gel (dessicant) - is used to absorb the water that is formed when hydrogen combines with the free oxygen in the presence of the catalyst. 4. Methylene blue or reazurin - is an oxygen reduction indicator that becomes colorless in the absence of oxygen.  ANAEROBIC CULTIVATION Anaerobic Incubator  When the CO₂ content of an  Its components are a dessicant, anaerobic incubator is increased to an oxygen-free indicator, a 10%, the oxygen is lowered to catalyst, and an anaerobic approximately 18%. source of gas (nitrogen, hydrogen, and carbon dioxide).  The ideal temperature to promote  The ideal anaerobic incubation growth of anaerobes is a 37 °C for 48 system is the anaerobic hours. chamber.  Isolation and susceptibility tests can also be performed within the chamber  ANAEROBIC CULTIVATION Gas-pak Jar  For a gas-pak jar, it takes 30 to 45  If the catalyst is working accurately, minutes to obtain an anaerobic water vapor will be present inside environment. the Gas-pak jar, and the indicator strip will be colorless.  When water is added to the gas-pak envelope, carbon dioxide and  It may take several hours for the hydrogen are produced. methylene blue indicator to change from blue to colorless.  BACTERIAL GROWTH CURVE The generation time of bacteria in a culture can be as brief as 20 minutes for a fast-growing bacterium such as Escherichia coli or as long as 24 hours for a slow-growing bacterium such as Mycobacterium tuberculosis.  BACTERIAL GROWTH CURVE Stages of Bacteria 2. Log or exponential phase 1. Lag phase or the period of (Balance growth) rejuvenescence  It is the period when  It is the period when there is microorganisms are actively no cell division or an abrupt growing and dividing. increase in the cell number.  It is the stage in which the bacteria increase logarithmically  It is the start of biosynthesis since cellular production is most although there is no increase active during this period. in cell mass.  It is the phase in which  It is the adjustment phase to a microorganisms are utilized in new environment. physiological and biochemical testing.  BACTERIAL GROWTH CURVE Stages of Bacteria 4. Death or decline phase 3. Stationary/Plateau phase  It is the period when there is a  It is the period when there is a balance between cell division and cessation of bacterial growth dying organisms, although the as the number of dead cells number of viable microorganisms exceeds the number of living remains constant. microorganisms.  It is the phase in which metabolic activities of surviving cells slow down  It is the stage in which there is and nutrients are becoming limited. a loss of nutrients and increase  It is the phase in which dead debris in the amount of toxic waste. starts to accumulate.  BACTERIAL GROWTH CURVE  Generation - is the doubling of the cell number.  Generation time/Doubling time - is the time required for bacteria to double their population.  METHODS FOR MEASURING BACTERIAL GROWTH 1. Microscopic Count  A measured volume of a bacterial suspension is placed inside a defined area on a microscope slide.  It does not distinguish between living and dead cells. 3. Turbidimetric Method- cell mass 2. Plate Count  Requires 10 to 100 million cells/mL  Most commonly used method  Measures the number of viable cells.  Determines the CPU/mL of bacteria  30 to 300 colonies should be counted.