Differential stain techniques Gram Staining

1. The differential stain technique distinguishes two kinds of organisms. An example is the Gram stain technique. 2. This differential technique separates bacteria into two groups, Gram-positive bacteria and Gram-negative bacteria. 3. Procedure Crystal violet is first applied, followed b! the mordant iodine, which fixes the stain . Then the slide is washed with alcohol, and the "ram#positi$e bacteria retain the cr!stal#$iolet iodine stain% howe$er, the "ram#negati$e bacteria lose the stain. The "ram#negati$e bacteria subsequentl! stain with the safranin dye, the counterstain, used next. These bacteria appear red under the oil#immersion lens, while "ram#positi$e bacteria appear blue or purple, reflecting the cr!stal $iolet retained during the washing step. The process canbe diagrammatically represented as follows :

Figure 1 The Gram stain procedure used for differentiating bacteria into two groups.

Acid ast Staining 1. Another differential stain technique is the acid-fast technique. This technique differentiates species of Mycobacterium from other bacteria. 2. &eat or a lipid sol$ent is used to carr! the first stain, carbolfuchsin, into the cells. Then the cells are washed with a dilute acid#alcohol solution. Mycobacterium species resist the effect of the acid#alcohol and retain the carbolfuchsin stain 'bright red(. 3. )ther bacteria lose the stain and take on the subsequent meth!lene blue stain 'blue(. Thus, the acid#fast bacteria appear bright red, while the nonacid#fast bacteria appear blue when obser$ed under oil#immersion microscop!.

Microorganisms Used in Alcohol Production There is a limited number of microorganisms which ferment carbohydrates (pentose or hexose sugars) into alcohols and yield some by-products. Microorganisms utilize various pathways. Following are some of alcohol producing micro-organisms !acteria Clostridium acetobutylicum, Klebsiella pneumoniae, Leuconostoc mesenteroides, Sarcina ventriculi, Zymomonas mobilis, etc. Fungi Aspergillus oryzae, Endomyces lactis, Kloeckera sp." Kluyreromyees fragilis, Mucor sp." eurospora crassa, !"izopus sp." Sacc"aromyces beticus, S# cerevisiae, S# elltpsoideus, S# oviformis, S# saki,$orula sp."$ric"osporium cutaneum, etc. # summary of alcohol production through different routes of microorganisms is given in Fig. $%.&.

i.

ii.

iii. iv. v.

Fermentable Substrates 'thyl alcohol is produced from such organic material that contains sugar or its precursor as fundamental units. The cost of substrates (raw materials) in fermentation is of ma(or consideration" because it directly affects the cost of products. The fermentable substrates used for alcohol production are given under )fermentable substrates) . !efore using in fermentation processes" the cellulosic" lignocellulosic and starchy materials are hydrolyzed by enzymes or acids (ust to render the complex substances into a simple forms (monosaccharides). 'nzymes for hydrolysis are obtained from barley malt or moulds by heat treatment of acidified materials. Klebsiella pneumonia is capable of utilizing the wood hemicellulose hydrolysate which consists of pentose and hexose sugars" low molecular weight oligomers and uronic acids. The products of fermentation are butanol" ethanol acetone" etc. These products can be utilized as solvent. Clostridium acetobutylicum anaerobically ferments the starchy substrates of grains and potatoes to produce acetone" ethanol and butanol. *ecently" it has been demonstrated that Sc"%aniomyces castellii directly converts the soluble starch into ethanol. Sugarcane molassess" a by- product of sugarcane mill" contains high percentage of sucrose and fructose sugars. The fruit (uice" for example grapes &'itis vinifera( contains high amount of sugars (+-$,- glucose and .-$/- fructose) when grapes are ripen. Moreover" percentage of sugar contents depends on ripeness of grapes. 0uices are obtained from the apples" pear"

palmyra and palm-flower stal1 to prepare alcoholic beverages sold in mar1et by different names in different countries. For instance" beverages named as wine" cider" perry and toddy are prepared from grapes" apple" pear and palmyra" respectively. 2imilarly" names of beverages prepared from different substrates are )3vass) (4.2.2.*.) and )!ear) (5ndia) from barley" )2onti)(5ndia) and 2a1e (0apan) from rice" )Merissa) (2udan) and )3affir bear) (Malawi)" from sorghum" )Thumba) (5ndia) and )!usa) (422*) from millets. Alcoholic Beverages and the microbes used in their preparations :

6ercentage of alcohol differs" in different alcoholic beverages . 2ome of them are briefly discussed i. Wine 5t is mainly an 'uropean drin1 produced from (uice of fresh grapes &'itis vinifera, '# rotundifolia(# 5n ripen grapes" concentration of sugar (glucose and fructose) increases. 7rape (uice (/.- sugar) is fermented by various strains of S# ellipsoideus into alcohol and also renders the chemical constituents which alters the flavor. ii. Beer !eer is produced after the fermentation of mixture of barley malt and starchy solutions by 2. cerevisiae (a top fermenter yeast that do not settle at bottom) or S# carlsbergensis (the bottom fermentation yeast).Malt is prepared from barley. 7rains are allowed to germinate. #fter &-8 days amylase and protease are formed. The sprouted grains are gradually heated to about +9:;. The dried rootlets are 1noc1ed off and the remaining grain is coarsely ground. Malt is mixed with coarsely ground starchy cereals (rice" maize" wheat) to produce grist. Mash is prepared by adding hot water to the grist holding for a period to allow enzymatic conversion and draining off the resultant sweet wart. <art is filtered and then boiled. Finally it is fermented with yeast . #fter fermentation sugar is converted into alcohol and also brings about minor chemical changes" for example protein. iii. Rum *um is the distilled product of culture fluid. S# cerevisiae or other yeast is used as the fermenting microorganism. ;ulture medium is prepared from blac1 strap molasses containing $/-$&- fermentable sugar. #mmonium sulphate and some times phosphates are added as nutrients. <hen fermentation is over" culture fluid is distilled to remove the alcohol and used as rum. iv. Whiskey 5t is prepared through the fermentation of grain mash (coo1ed and saccharified with peated malt) by a top yeast &S# cerevisiae( when fermentation is over" the culture fluid contains alcohols" traces of acids and esters v. Sake 2a1e" the rice wine" is manufactured from the strach. 5t is a complicated process which implies the mastering of different fermentation techni=ues in semi-solid and sub merged conditions" and regulation of successive microbial populations. First of all a mould &Aspergillus oryzae( then bacteria&Lactobacillus and Leuconostoc( and finally yeast (%. cerevisiae( are mixed with the fermentable medium. The culture fluid contains about /9- alcohol> therefore" before mar1eting the concentration of alcohol is ad(usted to $8- (2asson" $?+&).

Uses of Alcohols &a( Ethanol is used as solvent" extractant and antifreeze. 5t is also used as a substrate for the synthesis of many other solvents of dyes" 6harmaceuticals" lubricants" detergents" pesticides" plasticizers" explosives and resins" and for the manufacture of synthetic fibers .

&b) N-butanol is used in the manufacture of plasticizers" bra1e fluids" urea-formaldehyde resins" extractants and pertrol additives. &c( l!cerol is used in medicals" biosynthesis of @-fructose via mannitol" and in food industry (because of its sweetness and high solubility). Mannitol is used in industry and research. &d) Butanol plus acetone acid "# $-butanediol are used as industrial solvent. !utanol plus acetone is used in the production of explosive materials &e#g# cordite) and /" ,-butanediol in the synthesis of rubber.

&e( Ethanol is used as alcoholic beverages

$. #ntibiotics are chemicals that 1ill or inhibit the growth of bacteria and are used to treat bacterial infections. They are produced in nature by soil bacteria and fungi. This gives the microbe an advantage when competing for food and water and other limited resources in a particular habitat" as the antibiotic 1ills off their competition. /. #ntibiotics ta1e advantage of the difference between the structure of the bacterial cell and the hostAs cell. ,. They either prevent the bacterial cells from multiplying so that the bacterial population remains the same" allowing the hostAs defence mechanism to fight the infection or 1ill the bacteria" for example stopping the mechanism responsible for building their cell walls. &. #n antibiotic can also be classified according to the range of pathogens against which it is effective. 6enicillin 7 will destroy only a few species of bacteria and is 1nown as a narrow spectrum antibiotic. Tetracycline is effective against a wide range of organisms and is 1nown as a broad spectrum antibiotic. %. Penicillin

a. The antibacterial effect of penicillin was discovered by #lexander Fleming in $?/?. Be noted that
a fungal colony had grown as a contaminant on an agar plate strea1ed with the bacterium Stap"ylococcus aureus" and that the bacterial colonies around the fungus were transparent" because their cells were lysing. b. Be then recognised the importance of a fungal metabolite that might be used to control bacteria. The substance was named penicillin" because the fungal contaminant was identified as Penicillium notatum. Fleming found that it was effective against man! ram positive bacteria and he even used locally applied" crude preparations of this substance" from culture filtrates" to control eye infections. Cater on the product was purified by two other !ritish scientists" Florey and ;hain" wor1ing in the 42#" who managed to produce the antibiotic on an industrial scale for widespread use. #ll three scientists shared the Dobel 6rize for this wor1" and rightly so penicillin rapidly became the E%onder drugE which saved millions of lives. 5t is still a Efront lineE antibiotic" in common use for some bacterial infections although the development of penicillinresistance in several pathogenic bacteria now limits its effectiveness. c. The action of penicillin

This shows an )overlay plate)" in which a central colony of the fungus )enicillium notatum was allowed to grow on agar for %-8 days" then the plate was overlaid with a thin film of molten agar containing cells of the yellow bacterium" Micrococcus luteus. The production of penicillin by the fungus has created a zone of

growth inhibition of the bacterium. This demonstration parallels what #lexander Fleming would have observed originally" although he saw inhibition and cellular lysis of the bacterium Stap"ylococcus aureus. d. #n expanded role for the penicillins came from the discovery that natural penicillins can be modified chemically by removing the acyl group to leave &-aminopenicillanic acid (see diagram above) and then adding acyl groups that confer new properties.

e.

These modern semi-s!nthetic penicillins such as #mpicillin" ;arbenicillin and Fxacillin have various specific properties such as resistance to stomach acids so that they can be ta1en orally" a degree of resistance to penicillinase (a penicillin-destroying enzyme produced by some bacteria) extended range of activity against some 7ram-negative bacteria.

2ome examples of clinically important antibiotics produced by microbes are given as follows

2ome clinically important antibiotics Antibiotic 6enicillin ;ephalosporin 7riseofulvin !acitracin 6olymyxin ! #mphotericin Producer organism )enicillium c"rysogenum Cep"alosporium acremonium )enicillium griseofulvum *acillus subtilis *acillus polymy+a Streptomyces nodosus Activit! 7ram-positive bacteria !road spectrum @ermatophytic fungi 7ram-positive bacteria 7ram-negative bacteria Fungi Site or mode of action <all synthesis <all synthesis Microtubules <all synthesis ;ell membrane ;ell membrane

! 'rythromycin Deomycin 2treptomycin Tetracycline Gancomycin 7entamicin *ifamycin Streptomyces eryt"reus Streptomyces fradiae Streptomyces griseus Streptomyces rimosus Streptomyces orientalis Micromonospora purpurea Streptomyces mediterranei 7ram-positive bacteria !road spectrum 7ram-negative bacteria !road spectrum 7ram-positive bacteria !road spectrum Tuberculosis 6rotein synthesis 6rotein synthesis 6rotein synthesis 6rotein synthesis 6rotein synthesis 6rotein synthesis 6rotein synthesis

$. Frganic acids are valuable commodity chemicals or may be further processed into higher value chemicals" solvents" fuels" H bio-products. /. !io-based acids are produced through the fermentation of biomass sugars by a number of different microorganisms under different conditions" metabolic pathways" and efficiencies. ,. # variety of organisms in the production of organic acids and solvents through the fermentation of lactose" xylose" arabinose" H glucose. &. The process involves developing and improving bacterial microorganism strains" fermentation systems" bio-reactor designs" product recovery H separation systems for the production of organic acids and bio-solvents in non-sterile industrial environments. %. 5improvements of the process mainly focus on increased conversion rates H efficiencies" higher product concentrations" more efficient product recovery" and increasing reactor infection resistance. 8. The production of different organic acids (acetic acid" lactic acid" glucconic acid" succinic acid" etc.) through fermentation of biomass sugars can be a primary or secondary co-product in a biorefining system. Frganic acids can be produced and sold as commodity chemicals or further processed into higher value chemicals" biofuels" or bio-products. Advantages ;onversion of %-;arbon 2ugars ,ylose - Ara.binose" Bigher Metabolic ;onversion 'fficiency H *ates" 5ncreased 6roduct @iversity" *evenues" H 6rofitability"


3.

Bigher !iomass to 6roduct Iield H 6roduct Iield *ates"

*ncreased Product +oncentrations in ,ermentation, -ecreased .nerg! /sage in 0eparations 1 Product 2eco$er!, -ecreased ,ermentation *nfection 1 /psets, ,ermentati$e production of a number of organic acids% lactic, acetic, gluconic, citric, succinic, propionic, & butyric acids, from biomass deri$ed sugars through the de$elopment of impro$ed processes, bio#reactor s!stems, and organisms for the processing and reco$er! of $arious of organic acids, bio#sol$ents, and chemical precursors.

'hat are Biopesticides( ive some e)amples of microbial pesticides* !iopesticides are certain types of pesticides derived from such natural materials as animals" plants" bacteria" and certain minerals. For example" canola oil and ba1ing soda have pesticidal applications and are considered biopesticides.!iopesticides fall into three ma(or classes $. Microbial pesticides They consist of a microorganism (e.g." a bacterium" fungus" virus or protozoan) as the active ingredient. Microbial pesticides can control many different 1inds of pests" although each separate active ingredient is relatively specific for its target pestJsK. For example" there are fungi that control certain weeds" and other fungi that 1ill specific insects.The most widely used microbial pesticides are subspecies and strains of !acillus thuringiensis" or !t. /. Plant-+ncorporated-Protectants ,P+Ps- They are pesticidal substances that plants produce from genetic material that has been added to the plant. For example" scientists can ta1e the gene for the !t pesticidal protein" and introduce the gene into the plant)s own genetic material. Then the plant" instead of the !t bacterium" manufactures the substance that destroys the pest. The protein and its genetic material" but not the plant itself" are regulated by '6#. ,. Biochemical pesticides They are naturally occurring substances that control pests by non-toxic mechanisms. ;onventional pesticides" by contrast" are generally synthetic materials that directly 1ill or inactivate the pest. !iochemical pesticides include substances" such as insect sex pheromones" that interfere with mating" as well as various scented plant extracts that attract insect pests to traps. !ecause it is sometimes difficult to determine whether a substance meets the criteria for classification as a biochemical pesticide" '6# has established a special committee to ma1e such decisions. 2ome examples of biopesticides are as follows a. .iruses have been developed against insect pests such as Cepidoptera (butterflies and moths)" Bymenoptera (bees" wasps" and ants)" and @ipterans (flies).7ypsymoths and tent caterpillars" for example" periodically suffer fromepidemic virus infestations" which could be exploited and encouraged. b. Many commensal microorganisms (microorganisms that live on or in other organisms causing no direct benefit or harm) that occur on plant roots and leaves can passively protect plants against microbial pests by competitive exclusion (that is" simply crowding them out). *acillus

cereus has been used as an inoculumon soybean seeds to prevent infection by fungal pathogens in the genus ;ercospora. c. 2ome microorganisms used as biopesticides produce antibiotics" but the ma(or mechanism in most cases seems to be competitive exclusion. For example" Agrobacterium radiobacter antagonizes Agrobacterium tumefaciens" which causes the disease crown gall. d. 2pecies of two bacterial generaL*acillus and StreptomycesLwhen added as biopesticides to soil help control the damping-off disease of cucumbers" peas" and lettuce caused by *hizoctonia solani. !acillus subtilis added to plant tissue also controls stem rot and wilt rot caused by species of the fungus Fusarium. e. Mycobacteria species produce cellulose degrading enzymes" and their addition to young seedlings helps control fungal infection by species of 6ythium"*hizoctonia" and Fusarium. 2pecies of !acillus and 6seudomonas produce enzymes that dissolve fungal cell walls.

'hat are the advantages of using biopesticides( !iopesticides are usually inherently less to)ic than conventional pesticides. !iopesticides generally affect only the target pest and closely related organisms" in contrast to broad spectrum" conventional pesticides that may affect organisms as different as birds" insects" and mammals. !iopesticides often are effective in ver! small /uantities and often decompose =uic1ly" thereby resulting in lower exposures and largely avoiding the pollution problems caused by conventional pesticides. <hen used as a component of +ntegrated Pest Management ,+PM- programs " biopesticides can greatly decrease the use of conventional pesticides" while crop yields remain high .

Man! plants and animals are protected from pests b! passive means . For example" plant rotation is a traditional method of insect and disease protection that is achieved by removing the host plant long enough to reduce a regionAs pathogen and pest populations.

!iopesticides have several significant advantages over commercial pesticides. They appear to be ecologicall! safer than commercial pesticides because they do not accumulate in the food chain.

2ome biopesticides provide persistent control# as more than a single mutation is re=uired to adapt to them and because they can become an integral part of a pestAs life cycle. 5n addition" biopesticides have slight effects on ecological balances because they do not affect nontarget species.

Finally" biopesticides are compatible %ith other control agents.

Bacillus thuringiensis as biopesticide : E)plain* The most widely used microbial pesticides are subspecies and strains of !acillus thuringiensis" or !t. 'ach strain of this bacterium produces a different mix of proteins" and specifically 1ills one or a few related species of insect larvae. <hile some !t)s control moth larvae found on plants" other !t)s are specific for larvae of flies and mos=uitoes. The target insect species are determined by whether the particular !t produces a protein that can bind to a larval gut receptor" thereby causing the insect larvae to starve.

$. The best examples of microbial insecticides are *acillus t"uringiensis (!.t.) toxins" which were first used in $?9$. They have had widespread commercial production and use since the $?89As and have been successfully tested on $&9 insects" including mos=uitoes.

2. 5nsecticidal endotoxins are produced by !.t. during sporulation" and exotoxins are contained in
crystalline parasporal protein bodies. These protein crystals are insoluble in water but readily dissolve in an insectAs gut. ,. Fnce dissolved" the proteolytic enzymes paralyze the gut. 2pores that have been consumed germinate and 1ill the insect. !acillus popilliae is a related bacterium that produces an insecticidal spore that has been used to control 0apanese beetles" a corn pest. 4. The gene for the !.t. toxin has also been inserted into the genomes of cotton and corn" producing geneticall! modified# or M# plants that produce their own !.t. toxin. 7M cotton and !.t. corn both express the gene in their roots" which provides them with protection from root worms. %. 'cologists and environmentalists have expressed concern that constantly exposing pests to !.t. will cause insects to develop resistance to the toxin. 5n such a scenario" the effectiveness of traditionally applied !.t.would decrease.

there are se$eral characteristics that all culture media ha$e in common Media must be prepared in such a way that it is sterile prior to being inoculated with a bacterial sample" so that when a particular type of bacteria is cultured (cultivated) on that medium" it is the only type of bacteria present. 7rowth media must also provide everything the bacterial culture needs to live and grow" including water" nutrients" and the proper pB. Media can be either li=uid (nutrient broth) or solid (agar). Defined !edia versus Comple" !edia 0ome media formulations are $er! specific recipes in which certain ingredients must be present in specific amounts. These defined media 'also known as s!nthetic media( are used to grow bacteria that ha$e $er! particular needs. 4ost clinical cultures do not ha$e such exacting requirements, and can be grown in what is referred to as 5complex media6. +omplex media are composed of partiall! digested !east, beef, so! and additional proteins, in which the exact concentration and composition is unknown. *n comparison with defined media, which are good for growing bacteria with $er! particular needs, complex media can be thought of as a crowd#pleaser, suitable for growing man! different t!pes of less fastidious microbes.

*n addition to growth media formulations being classified as either defined or complex, there are also media that are designed to do more than 7ust grow bacteria, selecti$e and differential media pro$ide information about the bacteria growing. Selective #acterial Growth !edia 0electi$e media contain ingredients that inhibit the growth of certain t!pes of bacteria and8or encourage the growth of others. This t!pe of media is useful in helping to identif! unknown bacteria and in encouraging the growth of onl! the t!pes of bacteria that the microbiologist is interested in culti$ating. Differential #acterial Growth !edia -ifferential culture media are formulated to displa! a color change when the bacteria growing metaboli9e a certain ingredient. ,or example 4ac+onke!:s Agar, in addition to being selecti$e, contains the sugar lactose and a p& sensiti$e d!e. ;hen bacteria growing on 4A+ ferment lactose 'metaboli9e it for food(, the! generate acidic waste products that trigger the p& sensiti$e d!e to turn the bacteria pink. 0o, when grown on 4A+, colonies of "ram#negati$e, lactose fermenting bacteria are pink, the intensit! of the pink color corresponds to how good the bacteria are at eating lactose. +olonies of "ram#negati$e non# lactose fermenting bacteria grow in colorless colonies. Mannitol Salt Agar also contain food 'mannitol, a sugar alcohol( and a p& sensiti$e d!e. ;hen the bacteria growing on 40A ferment mannitol, the medium changes from its original pink color to a bright highlighter !ellow. <acteria that grow on 40A are all halophiles. *f halophilic normal flora bacteria are growing on 40A 'such as Staphylococcus epidermidis( the medium remains pink. *f halophilic pathogens are present 'such as Staphylococcus aureus(, the medium changes to bright !ellow. Another speciali9ed medium, <lood Agar '<AP( contains sheep:s blood, if bacteria growing on the medium produce exotoxins that hemol!9e 'cut up( the red blood cells, the medium changes color. <AP medium that has changed from red to transparent =completel! clear( indicates pathogen Staphylococcus pyogenes. <AP bruised or unaffected b! bacterial growth present indicate normal flora, non#pathogenic bacteria. ;ant to see more 0peciali9ed 4edia >isuals? ,or example 4ac+onke!:s Agar '4A+( is used to culti$ate "ram#negati$e bacteria, b! discouraging the growth of "ram positi$e bacteria through the use of cr!stal $iolet d!es and bile salts. Another selecti$e medium, 4annitol 0alt Agar '40A(, has a high concentration of sodium chloride, which selects for halophiles 'salt#lo$ing bacteria( such as members of the genus Staphylococcus.

Classification based on consistency: Culture media are liquid, semi-solid or solid. Liquid media are sometimes referred as broths (e.g nutrient broth). Liquid media are available for use in test-tubes, bottles or flasks. In liquid medium, bacteria grow uniformly producing general turbidity. Certain aerobic bacteria and those containing fimbriae (Vibrio & Bacillus) are known to grow as a thin film called surface pellicle on the surface of undisturbed broth. Bacillus anthracis is known to produce stalactite growth on ghee containing broth. Sometimes the initial turbidity may be followed by clearing due to autolysis, which is seen in penumococci. Long chains of Streptococci when grown in liquid media tend to entangle and settle to the bottom forming granular deposits but with a clear medium. Culturing bacteria in liquid media has some drawbacks. Properties of bacteria are not visible in liquid media and presence of more than one type of bacteria can not be detected. Liquid media tend to be used when a large number of bacteria have to be grown. Culture media are suitable to grow bacteria when the numbers in the inoculum is suspected to be low. Inoculating in the liquid medium also helps to dilute any inhibitors of bacterial growth. This is the practical approach in blood cultures. Culturing in liquid medium can be used to obtain viable count (dilution methods).

Solid media: Any liquid medium can be rendered by the addition of certain solidifying agents. Agar agar (simply called agar) is the most commonly used solidifying agent. The word "agar" comes from the Malay word agar agar (meaning jelly). It is also known as kanten, China grass, or Japanese isinglass. Agar is chiefly used as an ingredient in desserts throughout Japan. It is an unbranched polysaccharide obtained from the cell membranes of some species of red algae such as the genera Gelidium and Gracilaria, or seaweed (Sphaerococcus euchema). Commercially it is derived primarily from Gelidium amansii. Agar is composed of two long-chain polysaccharides (70% agarose and 30% agarapectin). It melts at 95oC (sol) and solidifies at 42oC (gel), doesnt contribute any nutritive property, it is not hydrolysed by most bacteria and is usually free from growth promoting or growth retarding substances. However, it may be a source of calcium & organic ions. Most commonly, it is used at concentration of 1-3% to make a solid agar medium. New Zealand agar has more gelling capacity than the Japanese agar. Agar is available as fibres (shreds) or as powders. For preparing agar in Petri plates, 3% agar (by weight) is added to the broth and autoclaved, when the medium is at ~50oC, it is poured on to sterile Petri plates and allowed to set. For preparing agar containing media in test-tubes, the culture medium is mixed with 3% agar and heated with stirring to melt. This ensures that all the tubes get equal amounts of agar. These tubes can then be sterilized by autoclaving. Semi-solid media Reducing the amount of agar to 0.2-0.5% renders a medium semi-solid. Such media are fairly soft and are useful in demonstrating bacterial motility and separating motile from non-motile strains (U-tube and Cragies tube). Certain transport media such as Stuarts and Amies media are semi-solid in consistency. Hugh & Leifsons oxidation fermentation test medium as well as mannitol motility medium are also semi-solid. Biphasic media Sometimes, a culture system comprises of both liquid and solid medium in the same bottle. This is known as biphasic medium (Castaneda system for blood culture). The inoculum is added to the liquid medium and when subcultures are to be made, the bottle is simply tilted to allow the liquid to flow over the solid medium. This obviates the need for frequent opening of the culture bottle to subculture. Preparation and storage: Care must be taken to adjust the pH of the medium before autoclaving. Various pH indicators that are in use include phenol red, neutral red, bromothymol blue, bromocresol purple etc. Dehydrated media are commercially available and must be reconstituted as per manufacturers recommendation. Most culture media are sterililized by autoclaving. Certain media that contain heat labile components like glucose, antibiotics, urea, serum, blood are not autoclaved. These components are filtered and may be added separately after the medium is autoclaved. Certain highly selective media such as Wilson and Blairs medium and TCBS agar need not be sterilized. It is imperative that a representation from each lot be tested for performance and contamination before use. Once prepared, media may be held at 4-5oC in the refrigerator for 1-2 weeks. Certain liquid media in screw capped bottles or tubes or cotton plugged can be held at room temperature for weeks.

Classification based on nutritional component: Media can be classified as simple, complex and synthetic (or defined). While most of the nutritional components are constant across various media, some bacteria need extra nutrients. Simple Media : Those bacteria that are able to grow with minimal requirements are said to nonfastidious and those that require extra nutrients are said to be fastidious. Simple media such as peptone water, nutrient agar can support most non-fastidious bacteria. Complex media such as blood agar have ingredients whose exact components are difficult to estimate.

Synthetic or defined media such as Davis & Mingioli medium are specially prepared media for research purposes where the composition of every component is well known. Classification based on functional use or application: These include basal media, enriched media, selective/enrichment media, indicator/differential media, transport media and holding media.

Basal Medium : Basal media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient broth and nutrient agar considered basal medium Enriched Medium : Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them enriched media. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Blood agar, chocolate agar, Loefflers serum slope etc are few of the enriched media.

Example : Blood agar is preparing by adding 5-10% (by volume) to a basal medium such as nutrient agar or other blood agar bases. Since blood can not be sterilized, it has to be collected aseptically from the animal. Animals have to be bled and the blood is collected in sterile containers with anticoagulant or glass beads. While sheep blood is preferred, blood from rabbit, horse and ox can also be collected. Human blood must be avoided since it may contain inhibitory substances including antibiotics. After the blood agar base is autoclaved, blood is added to the medium at temperature just above the solidifying point of agar. The mixture is then poured on to the plates and allowed to solidify. Blood agar is useful in demonstrating hemolytic properties of certain bacteria. Two major types of hemolysis are often seen on blood agar; beta and alpha hemolysis. Beta hemolysis is the complete lysis of RBC resulting in clearing around the colonies whereas alpha hemolysis is the partial lysis of RBC resulting in greenish discolouration around the colonies. Gamma hemolysis is a misnomer and it indicates non-hemolytic colonies. Chocolate agar is also known as heated blood agar or lysed blood agar. The procedure is similar to that of blood agar preparation except that the blood is added while the molten blood agar base is still hot. This lyses the blood cells and releases their contents into the medium. This process turns the medium brown, hence the name. This medium is especially useful in growing Hemophilus and Neisseria. Serum for medium can be obtained from animal blood but must be filtered through membrane or seitz filter before use. Selective and enrichment media are designed to inhibit unwanted commensal or contaminating bacteria and help to recover pathogen from a mixture of bacteria. While selective media are agar based, enrichment media are liquid in consistency. Both these media serve the same purpose. Any agar media can be made selective by addition of certain inhibitory agents that dont affect the pathogen. Various approaches to make a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these. Thayer Martin Agar used to recover N.gonorrhoeae contains Vancomycin, Colistin and Nystatin. Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contain 10% NaCl. Potassium tellurite medium used to recover C.diphtheriae contains 0.04% Potassium tellurite. McConkeys Agar used for Enterobacteriaceae members contains Bile salt that inhibits most gram positive bacteria. Pseudosel Agar (Cetrimide Agar) used to recover P.aeruginosa contains cetrimide. Crystal Violet Blood Agar used to recover S.pyogenes contains 0.0002% crystal violet. Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by incorporating Malachite green. Wilson & Blairs Agar for recovering S.typhi is rendered selective by the addition of dye Brilliant green. Selective media such as TCBS Agar and Monsurs Tellurite Taurocholate Gelatin Agar used for isolating V. cholerae from fecal specimens have elevated pH (8.5-5.6), which inhibits most other bacteria. Enrichment media are liquid media that also serves to inhibit commensals in the clinical specimen. Selenite F broth, tetrathionate broth and alkaline peptone water are used to recover pathogens from fecal specimens. Differential/Indicator media: Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony colour. Various approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that utilize them appear as differently coloured colonies. Such media are called differential media or indicator media. When a particular carbohydrate is incorporated

into a medium and a mixture of bacteria inoculated on it, only that bacterium that can ferment it produces acid. This change in pH is detected by using a pH indicator incorporated in the medium and the bacterium that can ferment the sugar appears in a different colour. This approach is used in MacConkeys agar, CLED agar, TCBS agar, XLD agar etc. MacConkeys agar is the most commonly used media to culture and identify gram negative bacilli (especially enterobacteriaceae members). It contains bile salts (selective agent), lactose (sugar), peptone and neutral red (pH indicator), agar and water. Those bacteria that can ferment lactose produce pink coloured colonies where non-lactose fermenting colonies produce colourless colonies. Similarly, Vibrio cholerae produces yellow coloured colonies on sucrose containing TCBS medium. Reduction of potassium tellurite to metallic tellurium by Corynebacterium diphtheriae results in production of black coloured colonies on PT agar. Production of H2S by Salmonella typhi results in production of black coloured colonies on Wilson & Blairs medium. Enterococcus fecalis produces black coloured colonies on bile esculin agar due to reduction of esculin to esculetin. Detection of hemolysis on blood agar can be considered as an indicator property of Blood agar. Transport media: Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms or commensals. This can be achieved by using transport media. Such media prevent drying (desiccation) of specimen, maintain the pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria. Some of these media (Stuarts & Amies) are semi-solid in consistency. Addition of charcoal serves to neutralize inhibitory factors. Cary Blair medium and Venkatraman Ramakrishnan medium are used to transport feces from suspected cholera patients. Sachs buffered glycerol saline is used to transport feces from patients suspected to be suffering from bacillary dysentery. Pikes medium is used to transport streptococci from throat specimens.

Anaerobic media: Anaerobic bacteria need special media for growth because they need low oxygen content, reduced oxidation reduction potential and extra nutrients. Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K. Such media may also have to be reduced by physical or chemical means. Boiling the medium serves to expel any dissolved oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can render a medium reduced. Robertson cooked meat that is commonly used to grow Clostridium spps medium contain a 2.5 cm column of bullock heart meat and 15 ml of nutrient broth. Before use the medium must be boiled in water bath to expel any dissolved oxygen and then sealed with sterile liquid paraffin. Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast extract and casein hydrolysate. Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in the medium. Under reduced condition, methylene blue is colourless.

Isolation of Pure Culture Though microorganisms are generally found in nature (air, soil and water) as mixed populations to study the specific role played by a specific microorganism in its environment, one must isolate the same in pure culture. Pure culture involves not only isolation of individual microorganisms from a mixed population, but also the maintenance of such individuals and their progenies in artificial media, where no other microorganisms find way to grow. However, it is not easy to isolate the individual microorganisms from natural habitats and grow them under imposed laboratory conditions. For this, great deal of laboratory manipulation is required. everal

methods for obtaining pure cultures are in use. ome common methods are in everyday!use by a ma"ority of microbiologists, while the others are methods used for special purposes. Common Methods of isolation of pure culture #ure culture of microorganisms that form discrete colonies on solid media, e.g., yeasts, most bacteria, many other microfungi, and unicellular microalgae, may be most commonly obtained by plating methods such as strea$ plate method, pour plate method and spread plate method. %ut, the microbes that have not yet been successfully cultivated on solid media and are cultivable only in liquid media are generally isolated by serial dilution method. 1 !trea" Plate Method This method is used most commonly to isolate pure cultures of bacteria. & small amount of mixed culture is placed on the tip of an inoculation loop'needle and is strea$ed across the surface of the agar medium. The successive strea$s (thin out( the inoculums sufficiently and the microorganisms are separated from each other. )t is usually advisable to strea$ out a second plate by the same loop'needle without reinoculation. These plates are incubated to allow the growth of colonies. The $ey principle of this method is that , by strea"ing, a dilution gradient is established across the face of the Petri plate as bacterial cells are deposited on the agar surface. %ecause of this dilution gradient, confluent growth does not ta$e place on that part of the medium where few bacterial cells are deposited
#arious methods of strea"ing

#resumably, each colony is the progeny of a single microbial cell thus representing a clone of pure culture. uch isolated colonies are pic$ed up separately using sterile inoculating loop' needle and restrea$ed onto fresh media to ensure purity. Pour Plate Method

This method involves plating of diluted samples mixed with melted agar medium. The main principle is to dilute the inoculum in successive tubes containing liquefied agar medium so as to permit a thorough distribution of bacterial cells within the medium. Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar medium maintained in the liquid state at a temperature of *+!*,-. (agar solidifies below *+-.). The bacteria and the melted medium are mixed well. The contents of each tube are poured into separate #etri plates, allowed to solidify, and then incubated. /hen bacterial colonies develop, one finds that isolated colonies develop both within the agar medium (subsurface colonies) and on the medium (surface colonies). These isolated colonies are then pic$ed up by inoculation loop and strea$ed onto another #etri plate to ensure purity. #our plate method has certain disadvantages as follows0 (i) the pic$ing up of subsurface colonies needs digging them out of the agar medium thus interfering with other colonies, and (ii the microbes being isolated must be able to withstand temporary exposure to the *+!*,- temperature of the liquid agar medium1 therefore this technique proves unsuitable for the isolation of psychrophilic microorganisms.

However, the pour plate method, in addition to its use in isolating pure cultures, is also used for determining the number of viable bacterial cells present in a culture. Pour Plate Method C. .olony development after incubation. &. #ouring of the plate1 .ontrol and consists of the sterili3ed plating medium alone

%. 2edia'dilution

The isolated colonies are pic$ed up and transferred onto fresh medium to ensure purity. )n contrast to pour plate method, only surface colonies develop in this method and the microorganisms are not required to withstand the temperature of the melted agar medium.

!pread Plate Method )n this method the mixed culture of microorganisms is not diluted in the melted agar medium (unli$e the pour plate method)1 it is rather diluted in a series of tubes containing sterile liquid, usually, water or physiological saline. & drop of so diluted liquid from each tube is placed on the centre of an agar plate and spread evenly over the surface by means of a sterili3ed bent!glass!rod. The medium is now incubated. /hen the colonies develop on the agar medium plates, it is found that there are some plates in which well!isolated colonies grow. This happens as a result of separation of individual microorganisms by spreading over the drop of diluted liquid on the medium of the plate. !erial (ilution Method &s stated earlier, this method is commonly used to obtain pure cultures of those microorganisms that have not yet been successfully cultivated on solid media and grow only in liquid media. & microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions. !pread plate method

'

The inoculum is sub"ected to serial dilution in a sterile liquid medium, and a large number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution. The aim of this dilution is to inoculate a series of tubes with a microbial suspension so dilute that there are some tubes showing growth of only one individual microbe. 4xample,if we have a culture containing 56 ml of liquid medium, containing 5,666 microorganisms i.e., 566 microorganisms'ml of the liquid medium. !erial dilution method

)f we ta$e out 5 ml of this medium and mix it with 7 ml of fresh sterile liquid medium, we would then have 566 microorganisms in 56 ml or 56 microorganisms' ml. )f we add 5 ml of this suspension to another 7 ml. of fresh sterile liquid medium, each ml would now contain a single microorganism. )f this tube shows any microbial growth, there is a very high probability that this growth has resulted from the introduction of a single microorganism in the medium and represents the pure culture of that microorganism.

!pecial Methods of Isolation on of Pure Culture 5. !ingle Cell Isolation methods &n individual cell of the required $ind is pic$ed out by this method from the mixed culture and is permitted to grow. The following two methods are in use.

(i) Capillary pipette method everal small drops of a suitably diluted culture medium are put on a sterile glass!coverslip by a sterile pipette drawn to a capillary. 8ne then examines each drop under the microscope until one finds such a drop, which contains only one microorganism. This drop is removed with a sterile capillary pipette to fresh medium. The individual microorganism present in the drop starts multiplying to yield a pure culture. (ii) Micromanipulator method 2icromanipulators have been built, which permit one to pic$ out a single cell from a mixed culture. This instrument is used in con"unction with a microscope to pic$ a single cell (particularly bacterial cell) from a hanging drop preparation. The micro!manipulator has micrometer ad"ustments by means of which its micropipette can be moved right and left, forward, and bac$ward, and up and down. & series of hanging drops of a diluted culture are placed on a special sterile coverslip by a micropipette. 9ow a hanging drop is searched, which contains only a single microorganism cell. This cell is drawn into the micropipette by gentle suction and then transferred to a large drop of sterile medium on another sterile coverslip. /hen the number of cells increases in that drop as a result of multiplication, the drop is transferred to a culture tube having suitable medium. This yields a pure culture of the required microorganism. The advantages of this method are that one can be reasonably sure that the cultures come from a single cell and one can obtain strains with in the species. The disadvantages are that the equipment is expensive, its manipulation is very tedious, and it requires a s$illed operator. This is the reason why this method is reserved for use in highly speciali3ed studies. +. )nrichment Culture Method :enerally, it is used to isolate those microorganisms, which are present in relatively small numbers or that have slow growth rates compared to the other species present in the mixed culture. The enrichment culture strategy provides a specially designed cultural environment by incorporating a specific nutrient in the medium and by modifying the physical conditions of the incubation. The medium of $nown composition and specific condition of incubation favors the growth of desired microorganisms but, is unsuitable for the growth of other types of microorganisms. Proof of Purity of Cultures &ssuming that one has isolated a pure culture, how does one establish that it is pure; & pure culture is one in which the cells are all of one $ind, i.e., demonstrate (li$eness(. Hence, the proof of purity of cultures consists of demonstrating the (li$eness( of microorganisms in the culture. )t is based on certain criteria as follows0 5.The microorganisms loo$ ali$e microscopically and stain in the same fashion. +. /hen plated, all the colonies formed loo$ ali$e.

<. trea$s, stabs, etc. are uniform. *. everal isolated colonies perform identically, i.e., ferment the same sugars, and so on. Capillary method for obtaining a single microbial cell

Maintenance and Preservation of Pure Cultures 8nce a microorganism has been isolated and grown in pure culture, it becomes necessary to maintain the viability and purity of the microorganism by $eeping the pure cultures free from contamination. 9ormally in laboratories, the pure cultures are transferred periodically onto or into a fresh medium (subculturing) to allow continuous growth and viability of microorganisms. The transfer is always sub"ect to aseptic conditions to avoid contamination. ince repeated sub culturing is time consuming, it becomes difficult to maintain a large number of pure cultures successfully for a long time. )n addition, there is a ris$ of genetic changes as well as contamination. Therefore, it is now being replaced by some modern methods that do not need frequent subculturing. These methods include refrigeration, paraffin method, cryopreservation, and lyophili3ation (free3e drying). *efrigeration #ure cultures can be successfully stored at 6!*-. either in refrigerators or in cold!rooms. This method is applied for short duration (+!< wee$s for bacteria and <!* months for fungi) because the metabolic activities of the microorganisms are greatly slowed down but not stopped. Thus their growth continues slowly, nutrients are utili3ed and waste products released in medium. This results in, finally, the death of the microbes after sometime. Paraffin Method This is a simple and most economical method of maintaining pure cultures of bacteria and fungi. )n this method, sterile liquid paraffin in poured over the slant (slope) of culture and stored upright at room temperature. The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium. This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture is preserved for several years. Cryopreservation .ryopreservation (i.e., free3ing in liquid nitrogen at !57=-.) helps survival of pure cultures for long storage times. )n this method, the microorganisms of culture are rapidly fro3en in liquid nitrogen at !57=-. in the presence of stabili3ing agents such as glycerol that prevent the formation of ice crystals and

promote cell survival. +yophili,ation (Free3e!>rying) )n this method, the culture is rapidly fro3en at a very low temperature (!?6-.) and then dehydrated by vacuum. @nder these conditions, the microbial cells are dehydrated and their metabolic activities are stopped1 as a result, the microbes go into dormant state and retain viability for years. Ayophili3ed or free3e!dried pure cultures and then sealed and stored in the dar$ at *-. in refrigerators. Free3e!drying method is the most frequently used technique by culture collection centers.