SELMA ABDUL SAMAD BCH-10-05-02 S1-MSc.BIOCHEMISTRY BACTERIAL CULTIVATION
Nutrients are substances used in biosynthesis and energy production. Hence required for bacterial growth Macronutrients (constitute 95 % of dry cell weight) C,O,H,N,S,P – Components of carbohydrates,lipids,proteins,nucleic acids K, Ca,Mg,Fe– Exist as cations with different roles – eg.,inenzymes,heat resistance to endospores, cofactors, cytochromes etc. NUTRITIONAL REQUIREMENTS OF BACTERIA
Micronutrients – Mn,Zn,Co,Mb,Ni,Cu – As part of enzymes and cofactors Special requirements – eg.- Diatoms need silicic acid(H4SiO4) to construct their silica cell wall eg.- Halophytes need more sodium All nutrients should be present balanced
Usually satisfied from same source Usual sources for carbon are CO2 for autotrophs(from atmosphere or cellular metabolism) Glucose for heterotrophs Most bacteria – flexible in case of carbon source REQUIREMENTS FOR C , H , O
Based on oxygen requirement – aerobic and non-aerobic Aerobes – require O2 for growth Obligate aerobes- eg.vibriocholerae Facultative aerobes – most medically important bacteria Anaerobes- eg. Clostridia Obligate- die on oxygen exposure Facultative- grow well in anaerobic conditions Microaerophilic- grow well in low oxygen tension
Nitrogen – for synthesis of amino acids,nitrogenousbases,some carbohydrates and lipids,enzyme cofactors etc SOURCE – aminoacids,ammonia,nitrates(to NH3 by phototrophs),nitrogen(N2 fixation by rhizobium) Phosphorous – nucleic acids,phospholipids,nucleotides,cofactors,proteins,cell components SOURCE-inorganic phosphate,organic P(eg.Hexose.6.P) Sulfur – aminoacids(cys,met),some carbohydrates,biotin,thiamine SOURCE- sulfates REQUIREMENTS FOR N , P , S
Organic components required because they are essential cell components or precursors of such components but cannot be synthesised by the organism are called growth factors For bacteria they are Amino acids Purines and pyrimidines vitamins GROWTH FACTORS
A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions. A culture of bacillus anthacis CULTURE MEDIA
A culture medium must supply suitable carbon and energy sources and other nutrients, sometimes including "growth factors" (defined below). It is important to note that no one medium will support the growth of all microorganisms Culture medium – a liquid/gel designed to support the growth of micro-organisms 2 common types – nutrient broth and nutrient agar Basically differentiated to Defined(known quantity of all ingredients) Undefined(has some complex ingredients)
C source – CO2(as Na2CO3 / bicarbonate) N source – nitrate / ammonia Also sulfate and phosphate Used for – photolithoautotrophs like cyanobacteria SYNTHETIC/DEFINED MEDIA
Undefined components like peptones, meat extract, yeast extract (each one serves as C, N and energy source) Eg. Nutrient broth, tryptic soy broth, MacConkey agar etc. COMPLEX/UNDEFINED MEDIA
SIMPLE/BASAL MEDIA Eg.Nutrient broth – peptone,meatextract,NaCl and water. Nutrient agar is 2% agar added to broth SELECTIVE MEDIA 0 Favor growth of a particular micro organism 0 bile salts/dyes(basic fuchsin/crystal violet) – favor growth of gram negative bacteria and inhibit gram positive bacteria 0 endo agar, EMB agar, MacConkey agar – contain dyes that suppress gram positive bacteria 0 Media with only cellulose as C and energy source – selects cellulose digesting bacteria OTHER TYPES OF MEDIA
ENRICHED MEDIA 0substances like blood ,serum or egg are added to basal media 0 Used to grow bacteria which are more exacting in their nutritional needs. 0 eg. Blood agar, chocolate agar , egg media ENRICHMENT MEDIA 0 Substances which have a stimulating effect on the bacteria to be grown or an inhibitory effect on those to be suppressed are incorporated in the media(usually liquid media) 0 An absolute increase in the number of the wanted bacterium relative to the other 0 eg. Tetrathionate broth (inhibits coliforms , allows typhoid – paratyphoid bacilli to grow freely 0 Selenite F broth – favors dysentery bacilli
DIFFERENTIAL MEDIA 0 Distinguishes between different groups of bacteria 0 Blood agar (also an enriched media) distinguish between hemolytic and non-hemolytic bacteria Hemolytic bacteria ( eg. Many streptococci and staphylococci isolated from throat ) produce clear zones around their colonies 0 MacConkey agar (also a selective media) since it contains lactose and neutral red dye , lactose fermenting colonies appear pink to red in color , thus getting distinguished.
Blood agar plates are often used to diagnose infection. On the right is a positive Streptococcus culture; on the left a positive Staphylococcus culture
Pure culture – population of cells arising from a single cell, to characterize an individual species Different methods are used SPREAD PLATE – A mixture of cells spread out on an agar surface – each cell grows into a colony – each colony represents a pure culture ,and the number of viable organisms. STREAK PLATE – A mixture of cells transferred to the edge of an agar plate with an inoculating loop and streaked out in a pattern ISOLATION OF PURE CULTURE
POUR PLATE The original sample is diluted several times – small volumes of diluted sample is mixed with liquid agar that has been cooled to about 45 degree , poured immediately into sterile culture dishes – each cell forms individual colony
STERILIZATION – The process by which an article, surface or medium is freed of all living microorganisms either in vegetative or spore state. DISINFECTION – The destruction or removal of all pathogenic organisms STERILISATION and DISINFECTION
PHYSICAL AGENTS Sunlight Drying Dry heat : flaming, incineration, hot air Moist heat : pasteurisation, boiling, steam under pressure, steam under normal pressure Filtration : candles, asbestos pads, membranes. Radiation Ultrasonic and sonic vibrations STERILISING AGENTS
CHEMICAL AGENTS Alcohols : ethyl, isopropyl, trichlorobutanol Aldehydes : formaldehyde, glutaraldehyde Dyes Halogens Phenols Surface active agents Metallic salts Gases : ethylene oxide, formaldehyde, beta propiolactone
SUNLIGHT Has UV rays that are sterilizing along with heat rays Bacteria suspended in water are readily destroyed by exposure to sunlight DRYING Moisture is essential for bacterial growth(4/5 of bacterial cell weight is of water) Spores are unaffected by drying HEAT Most reliable method of sterilization Influenced by nature (dry or moist) , temperature and time, type and characteristics of microorganisms present and which material to be sterilised Heating denatures protein,causes oxidative damage,toxicity due to elevated electrolytes etc.
DRY HEAT FLAMING – Inoculating loop or wire, the tip of forceps and searing spatulas are held in a bunsen flame till they become red hot. INCINERATION – Used for safely destroying contaminated materials – like cloth , animal carcasses and pathological materials HOT AIR OVEN – Most widely used method – used for instruments and glasswares, forceps, scissors ,scalpels etc. Also paraffin,dusting powder, fats and grease. The spores of a nontoxigenic strain of Clostridium tetani are used as a microiological test of dry heat efficiency.
MOIST HEAT PASTEURISATION – for milk – heated either at 63` for 30 min or 72` for 15-20 min All nonsporing pathogens are destroyed BOILING – vegetative bacteria are killed almost immediately at 90-100` C ,but sporing bacteria require prolonged periods of boiling. It is only a means for disinfection and not sterilisation STEAM UNDER PRESSURE – The principle of autoclave or steam steriliser is that water boils when its vapor pressure equals that of the surrounding atmosphere.Hence when pressure inside a closed vessel increases, the temperature at which water boils also increases. The condensed water ensures moist conditions for killing the microbes present
FILTRATION Helps to remove bacteria from heat labile liquids such as sera and solutions of sugars or antibiotics used for preparation of culture media RADIATION NON IONISING LOW ENERGY TYPE - electromagnetic rays with wavelengths longer than those of visible light are used. Also uv radiations are used IONISING HIGH ENERGY TYPE - x rays, gamma rays and cosmic rays are highly lethal to DNA and other vital constituents. VIBRATIONS Has no practical value in sterilisation and disinfection since micro organisms vary in their sensitivity to them
CHEMICAL AGENTS ALCOHOLS – ethyl alcohol and isopropyl alcohol are most frequently used. They have no action on spores Methyl alcohol is effective against fungal spores(hence used for treating cabinets and incubators) ALDEHYDES – Formaldehyde is active against the amino group in the protein molecule. It is bactericidal and sporicidal,also has lethal effect on virus Glutaraldehyde is also used and is less toxic than the former DYES – aniline dyes and acridine dyes are used as skin and wound antiseptics Both are just bacteriostatic and has low bactericidal activity. Aniline dyes(brilliant green,malachitegreen,crystal violet) against gram positive organisms.Same with acridine dyes too.
HALOGENS – iodine in aqueous and alcoholic solution has been used widely as a skin disinfectant. Actively bactericidal with moderate action against spores Chlorine and its compounds used as dinsinfectants in water supplies, swimming pools etc. PHENOLS – cause cell membrane damage ,releasing cell contents and causing lysis. Low concppt proteins. Membran bound oxidases and dehydrogenases are inactivated by conc of phenol that are rapidly bactericidal for microbes GASES – it alkylates the amino , carboxyl ,hydroxyl and sulfhydryl groups in protein molecules. In addition , it reacts with DNA and RNA. Its use as a disinfectant presents a potential toxicity to human beings, including mutagenicity and carcinogenicity. It’s effective against viruses and spores.
SURFACE ACTIVE AGENTS – Substances which alter energy relationship at interfaces, producing a reduction of surface or interfacial tension are referred to as surface active agents. They usually act on bacterial membranes Cationic agents act on the phosphate groups of the cell membrane and also enter the cell METALLIC SALTS – salts of heavy metals have a greater action. Salts of silver, copper and mercury are used as disinfectants They are protein coagulants and have the capacity to combine with free sulfhydryl groups of cell enzymes, when used at appropriate conc. Eg. Copper salts , phenyl mercury nitrate, and mercurochrome.