Microbial Detection and Staining Techniques PDF

Culture media An artificial culture media must provide environmental and nutritional conditions that exist in the natural habitat of a...

Culture media An artificial culture media must provide environmental and nutritional conditions that exist in the natural habitat of a bacterium. Microbial detection based on A culture medium contains water, a source of carbon & energy, source of nitrogen, trace elements (minerals) and bacterial structure; Stains & some growth factors. The pH of the medium must be set accordingly. staining techniques Uses: ✓ Enrich the number of bacteria ✓ Select for certain bacteria and suppress others ✓ Differentiate among different kinds of bacteria Cultivation/Culturing of Bacteria Pure culture In the laboratory bacteria are isolated and grown in A microbial culture, is a method of multiplying microorganisms by pure culture in order to study the functions of a letting them reproduce in predetermined culture media under controlled laboratory conditions. particular specie. Microbial cultures are used to determine the type of organism, its abundance in the sample being tested, or both. A pure culture is a population of cells or growing in the absence of other species or types. A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another. Pure culture Agar Pure cultures are obtained by using variety of special Agar, a polysaccharide extracted from marine algae, is used to techniques. All glassware, media and instruments must be solidify a specific nutrient solution. sterilized i.e. aseptic techniques are used for obtaining Unlike other gelling agent, it is not easily degraded by many pure cultures. bacteria. Basic requirement for obtaining a pure culture are solid It is not easily destroyed at higher temperatures, and medium, a media container that can be maintained in an therefore it can be sterilized by heating, the process which aseptic condition and a method to separate individual also liquefies it. cell. Once solidified, agar medium will remain solid. A single bacterium, supplied with right nutrients, will The culture media is contained in a petri dish, a two part, glass multiply on the solid medium in a limited area to form a or plastic covered container. colony, which is a mass of cells all descended from the original one. Aseptic techniques Classification of culture media Aseptic technique is a method that involves target-specific practices and procedures under suitably controlled Bacterial culture media can be classified in at least conditions to reduce the contamination from microbes.. three ways: 1- Consistency 2- Nutritional component 3- Functional use  Classification based on consistency Classification based on consistency 1- Liquid media 2- Solid media 3- Semi-solid media Classification based on consistency Classification based on Nutritional Components A. Liquid media: these are available for use in test- tubes, bottles or flasks. Liquid media are sometimes referred as “broths” (e.g. 1. Simple media. nutrient broth). In liquid medium, bacteria grow 2. Complex media. uniformly producing general turbidity. No agar is added. Mostly used for inoculums preparation. 3. Synthetic or chemically defined media. B. Solid media: an agar plate is a petri dish that contains a growth medium (typically agar plus nutrients) used to culture microorganisms. 2% of agar is added. Morphology, pigmentation, hemolysis can be appreciated. Examples include Nutrient agar and Blood agar. C. Semi-solid media: such media are fairly soft and are useful in demonstrating bacterial motility and separating motile from non-motile strains. Examples of semi-solid media (Hugh & Leifson’s oxidation fermentation). 0.5% agar is added. Classification based on nutritional components Classification based on Functional Use or 1. Simple media: simple media such as peptone water, nutrient agar can Application support most non-fastidious bacteria. It is also called as basal media. Eg: NB, NA. nutrient broth consists of peptone, yeast extract and Nacl. When 2% of agar is added to Nutrient broth it forms nutrient agar. 1. Enriched media. 2. Selective media. 2. Complex media: media other than basal media are called complex media. They have special ingredients in them for the growth of 3. Differential media. microorganisms. These special ingredients like yeast extracts, consist 4. Transport media. of a mixture of many chemicals in an unknown proportion. 5. Indicator media. 6. Anaerobic media. 3. Synthetic media/Chemically defined media: specially prepared media for research purposes where the composition of every component is well known. It is prepared from pure chemical substances. Eg: peptone water (1% peptone + 0.5% NaCl in water). Peptone water is a microbial growth medium composed of peptic digest of animal tissue and sodium chloride. Fastidious vs Non-Fastidious bacteria Enriched media 1.Enriched media: Addition of extra-nutrients in the form of blood, serum, egg yolk etc. to basal medium makes them enriched media. Media used to isolate pathogens from a mixed culture. Stimulate growth of desired bacterium and inhibit growth of unwanted bacterium. Media contains components that suppress the unwanted organism, thus increase in numbers of desired bacteria. Examples of enriched media: Chocolate agar and blood agar. Selenite F Broth – for the isolation of salmonella, shigella. Tetrathionate Broth – inhibit coliforms. Alkaline peptone water- for Vibrio cholera.  Enriched media: Chocolate and blood agar Enriched media: Selenite broth and Alkaline peptone water Selenite F Broth: Selenite broth is a selective medium Chocolate agar: a non-selective enriched growth which is inhibitory to organisms such medium used for growing fastidious bacteria, such as E.coli and Enterococcus which are more sensitive as Haemophilus influenza. to the toxic effects of sodium selenite than are salmonellas. The selective effect is not complete, for although the unwanted species are inhibited during the Blood agar: Blood agar plate (BAP) contains first 12 hours of incubation, they later increase rapidly. mammalian blood ( usually sheep or horse), typically Salmonellas, in contrast, are able to multiply at a concentration of 5-10%. BAP are enriched, throughout, allowing enrichment to occur. differential media used to isolate fastidious organisms and detect hemolytic activity. Alkaline Peptone Water is a broth medium for the enrichment of Vibrio species from food, water and clinical samples. The alkaline pH of this medium allows the growth of Vibrio organisms while inhibiting the growth of commensal intestinal bacteria. Enriched media: Chocolate and blood agar Selective media The inhibitory substance is added to a solid media thus causing an increase in number of colonies of desired bacterium. Chocolate agar is prepared by heating blood agar, which in turn ruptures the red blood cell (RBC) and releases nutrients that aid in the Selective media and enrichment media are designed to inhibit unwanted growth of fastidious bacteria, most notably commensal or contaminating bacteria and help to recover pathogen from Haemophilus and Neisseria species. a mixture of bacteria. The name is derived from the fact that the Any agar media can be made selective by addition of certain inhibitory lysis of RBC gives the medium a chocolate- agents that don’t affect the pathogen. To make a medium selective brown color. include addition of antibiotics, dyes, chemicals, alteration of pH or a Chocolate agar (CHOC) or chocolate blood combination of these. agar (CBA), is a non-selective, enriched growth medium used for isolation of pathogenic bacteria. It is a variant of the blood agar plate, containing red blood cells that have been lysed by slowly heating to 80°C. Selective media: EMB agar Differential media: MacConkey agar Eosine Methylene Blue (EMB) agar is selective for Gram- negative bacteria. The dye methylene blue in the medium MacConkey agar inhibits the growth of gram-positive bacteria, small medium designed to amounts of this dye effectively inhibit the growth of most grow gram-negative gram-positive bacteria. bacteria and EMB is a microbiological medium, which slightly inhibits the differentiate them for growth of Gram-positive bacteria and provides a color lactose fermentation. indicator distinguishing between organisms that ferment lactose. Lactose fermenters- Organisms that ferment lactose display colonies with dark pink colonies- centers. and non lactose Rapid lactose fermentation produces acids, which lower fermenters- colorless the pH. This encourages dye absorption by the colonies, colonies-. which are now colored purple-black. This medium is important in medical laboratories by distinguishing pathogenic microbes in a short period of time. Differential Media Differential media: CLED agar Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony color. Cystine Lactose Electrolyte Deficient (CLED) Agar is used for the differentiation Various approaches include incorporation of dyes, metabolic substrates etc, and enumeration of microorganisms in so that those bacteria that utilize them appear as differently colored colonies. urine. It is also based on lactose Substances incorporated in it enable it to distinguish between bacteria. fermentation. A medium for urine culture where the absence of electrolytes inhibits Examples: MacConkey’s agar, CLED agar the swarming of Proteus spp. Cystine is added for the benefit of those organisms which have a specific cystine requirement. The key difference between selective media and differential media is that selective media are used to grow and isolate a specific type of Organism Colony Morphology microorganism by suppressing the growth of other deep yellow colonies about 0.75 mm microorganisms while differential media are used to visually distinguish Staphylococcus aureus diameter, uniform in color. yellow medium microorganisms from one another Corynebacteria very small grey colonies similar to corynebacteria but with a Lactobacilli rougher surface  Transport media Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms or commensals. Delicate organisms may not survive the time taken for transporting the specimen without a transport media. This can be achieved by using transport media. Transport media should fulfill the following criteria: - Temporary storage of specimens being transported to the laboratory for cultivation. - Maintain the viability of all organisms in the specimen without altering their concentration. - Contain only buffers and salts. - Lack of carbon, nitrogen, and organic growth factors so as to prevent microbial multiplication. - Transport media used in the isolation of anaerobes must be free of molecular oxygen. Difference between selective, Examples of Transport media differential and enriched media Cary Blair medium ( for campylobacter species) Sodium chloride: 5.0 Calcium chloride: 0.1 Sodium thioglycollate: 1.5 PH: 8.4 ± 0.2 @ 25°C Indicator media Anaerobic media: Thioglycollate Contains an indicator which changes its color when a bacterium grows in them. e.g: Urease media: Urea-> CO2 +NH3 NH3 -> medium turns pink Indicator: phenol red, medium becomes alkaline. Anaerobic media Culture methods - Anaerobic bacteria need special media for growth because they need low oxygen content, reduced oxidation- reduction potential; and extra nutrients. - Streak culture - Lawn culture - Media for anaerobes may have to be supplemented with - Stroke culture nutrients like hemin and vitamin K. - Stab culture - Boiling the medium serves to expel any dissolved oxygen. - Pour plate method Example of Anaerobic media: Thioglycollate medium  Streak culture Lawn culture Used for the isolation of bacteria in pure culture from clinical specimens. Provides a uniform surface growth of the bacterium. Platinum wire is used. One loop full of the specimen is transferred onto the surface of Lawn cultures are prepared by a well dried plate. flooding the surface of the plate Spread over a small area at the periphery. with a liquid suspension of the The inoculum is then distributed thinly over the plate by streaking bacterium. it with a loop in a series of parallel lines in different segments of the plate. Uses: On inoculation, separated colonies are obtained over the last - For bacteriophage typing series of streaks. - Antibiotic sensitivity testing - In the preparation of bacterial antigens and vaccines Streak Culture Stroke culture Stroke culture is made in tubes containing agar/ slant. An agar slant tube (or simply an agar slant) is a screw- capped culture tube partly filled with an agar mix. To make it a slant tube the agar is allowed to cool with the tube laying at an angle, resulting in a large surface area for spreading a culture Uses: Provides a pure growth of bacterium for slide agglutination and other diagnostic tests. Slants are better suited than agar plates because they can be capped, preventing the agar and culture from drying out. The cap also prevents airborne contaminates from entering the slant. Slants take up less storage space. Stab culture Pour plate culture Prepared by puncturing a suitable medium- gelatin or glucose agar with a long, straight, charged wire. Stab cultures are similar to agar plates, but are formed by solid agar in a test tube. Bacteria is introduced via an inoculation needle or a pipette tip being stabbed into the center of the agar. Bacteria grow in the punctured area. Stab cultures are most commonly used for short- term storage or shipment of cultures - Maintenance of stock cultures Pour plate culture Culture methods Pour plate method is usually the method of choice for counting the number of colony-forming bacteria present in a liquid specimen. Because the sample is mixed with the molten agar medium, a larger volume can be used than with the spread plate.... Each colony represents a “colony-forming unit” (CFU). 1 ml of the inoculum is added to the molten agar Mix well and pour to a sterile petri dish Uses: - Gives an estimate of the viable bacterial count in a suspension. - For the quantitative urine cultures  STAINS AND DYES REQUIREMENTS FOR STAINING A dye is a general-purpose coloring agent, whereas Stain – Majority of the stains used for staining bacteria are of the a stain is used for coloring biological material. basic type as nucleic acid of bacterial cells attract the positive ions, e.g. methylene blue, crystal violet. Acidic stains are used for background staining. A stain is an organic compound containing a Mordant – It is a chemical that forms an insoluble complex benzene ring plus a chromophore and an with the stain and fixes it or causes the stain to penetrate auxochrome group. more deeply into the cell. These are used in indirect staining. For example, Gram’s iodine in Gram staining and phenol in chromophore is a chemical group that imparts Ziehl Neelson’s staining. color to benzene. They form a chromogen. Accentuater – It is a chemical which when added to a stain to auxochrome group is a chemical compound that make the reaction more selective and intense. conveys the property of ionization of chromogen Decolorizer – It is a chemical used to remove the excess stain in (ability to form salts) and bind to fibers or tissues. indirect regressive staining. For example, ethanol in Gram’s staining. OBJECTIVES OF STAINING Types of staining techniques Improves visibiltiy by greater contrast between the organism and the background, Simple staining Differential staining differentiate various morphological types (by shape, size, arrangement, etc.), (Use of single stain) (Use of two contrasting stains) determine the staining characteristic of organism and, at times, direct diagnosis of disease, Direct Indirect Separation Visualization demonstrate the purity of culture. (Positive) (Negative) into groups of structures observe certain structures (flagella, capsules, endospores, etc.), 1. Gram stain 1. Flagella stain 2. Acid fast 2. Capsule stain stain 3. Spore stain SIMPLE STAINING Direct staining (Positive staining) A simple staining technique that stains the bacterial cells in a A staining method that uses only a single dye which single color. does not differentiate between different types of organisms Many of the bacterial stains are basic chemicals; these There is only a single staining step and everything is stained with the same color. basic dyes react with negatively charged bacterial cytoplasm (opposite charges attract) and the organism Simple stains are used to stain whole cells or to stain becomes directly stained. specific cellular components. Examples are methylene blue, crystal violet, and basic Types of simple staining: fuchsin. 1. Direct / Positive staining : stain object 2. Indirect / Negative staining: stain background Indirect staining (Negative staining) In this staining process, instead of cells, background is stained. Here, an acidic dye like nigrosin or Indian ink is used. Acidic stain carries a negative charge and repelled by the bacteria, which also carry a negative charge on their surface. Hence, an acidic dye do not stain bacteria. Instead, it forms a deposit around the organism, leaving the organism itself colorless or transparent upon examination.  STAINING: GENERAL TECHNIQUE DIFFERENTIAL STAINING SMEAR AIR DRY HEAT FIX STAIN LOOK A bacterial smear is a thin layer of bacteria placed on a slide for staining. Preparing the smear requires attention to a number of details that help prevent contamination of the culture. Importance of fixing the smears Fixation accomplishes three things: (1) it kills the organisms; (2) it causes the organisms to adhere to the slide; GRAM’S STAINING (3) it alters the organisms so that they more readily accept stains (dyes). The Gram Stain GRAM POSITIVE Lipoteichoic acid Peptidoglycan-teichoic acid In the late 1800’s, Christian Gram observed that some genera of bacteria retained a dye-Iodine complex when rinsed with alcohol, while other genera were easily decolorized with alcohol and could be then visualized by a Cytoplasmic membrane contrasting counter stain. Cytoplasm This staining procedure defines two bacterial groups: those GRAM NEGATIVE Porin Lipopolysaccharide which retain the primary dyes (“Positive by Gram’s Method” or “Gram-Positive”) and those which are easily Outer Membrane Braun lipoprotein decolorized (“Negative by Gram’s Method” or “Gram- Negative”). This is the starting point for bacterial Inner (cytoplasmic) membrane identification procedures. Cytoplasm The Gram Stain Gram staining – Requirements The difference in dye retention is dependent on such Gram-staining is a four part procedure. physical properties as thickness, density, porosity, and The specimen is mounted and heat fixed on a slide before integrity of the bacterial cell wall, as well as its proceeding to stain it. chemical composition. Gram-Positive bacteria have thick, dense, relatively non- The reagents required are: porous walls, while Gram-Negative bacteria have thin Crystal Violet (the Primary Stain) walls surrounded by lipid-rich membranes. Iodine Solution (the Mordant) Gram-Positive bacteria which have lost wall integrity Decolorizer (ethanol) through aging or physical or chemical damage may stain Safranin (the Counter stain) Gram-Negative. Water (preferably in a squirt bottle)  Gram staining – Procedure 1 The bacteria are first stained with the basic dye crystal violet (primary stain). Both gram-positive and gram-negative bacteria become directly stained and appear purple after this step. 2 The bacteria are then treated with gram's iodine solution (mordant). This allows the stain to be retained better by forming an insoluble crystal violet- iodine complex, called as ‘iodine lake’. Both gram-positive and gram- negative bacteria remain purple after this step. 3 Gram's decolorizer, a mixture of ethyl alcohol and acetone, is then added. This is the differential step. Gram-positive bacteria retain the crystal violet- iodine complex while gram-negative are decolorized. 4 Finally, the counter stain safranin (also a basic dye) is applied. Since the gram-positive bacteria are already stained purple, they are not affected by the counter stain. Gram-negative bacteria, that are now colorless, become directly stained by the safranin. Thus, gram-positive appear purple, and gram-negative appear red. ACID FAST STAINING Structure of an Acid-Fast Cell Wall Acid fast staining - theory Once stained the acid fast bacterial cells resist decolorization with acidified organic solvents, e.g acid alcohol and are therefore called ACID FAST. Acid fast staining property of the genus, Mycobacteria, depends upon their lipid-rich cell walls which are relatively impermeable to various basic dyes unless the dyes are combined with phenol. The exact method by which the stain is retained is unclear but it is thought that some of the stain becomes trapped within the cell and some forms a complex with the mycolic acids. This is supported by the finding that shorter chain mycolic acids or mycobacterial cells with disrupted cell walls stain weakly acid-fast, e.g. Nocardia Structure of an Acid-Fast Cell Wall Principle Stain used consists of basic fuchsin, with phenol added. Acid fast bacteria retain primary stain (carbol fuchsin) even after washing with a strong acid. It appears red while non- acid fast bacteria are decolorized on washing with acid and takes the color of the counter stain (methylene blue). The property of acid fasting appears due to the presence of mycolic acid in their cell walls. Mycolic acid is a group of branched chain hydroxy lipids. 64  Acid-Fast Stain of Mycobacterium tuberculosis in It is most commonly used to identify M.tuberculosis and M.leprae, the pathogen responsible for tuberculosis Sputum and leprosy, respectively. These bacteria have cell walls containing lipids constructed from mycolic acids, a group of branched chain hydroxyl fatty acids, which prevent dyes readily binding to the cells. Once basic fuchsin has penetrated, M.tuberculosis and M.leprae are not easily decolorized by acidified alcohol (acid-alcohol) and thus are said to be acid-fast. Non acid-fast bacteria are decolorized by acid-alcohol and thus are stained blue by methylene blue counterstain. Note the reddish acid-fast bacilli among the blue normal flora and 65 white blood cells in the sputum that are not acid-fast. Procedure SPECIAL STAINING 66 CAPSULE STAINING Some bacteria secrete a prominent slimy or gummy material on their surface usually polysaccharide make up the capsule. It is not essential for life, but provides protection against dehydration and also against phagocytosis. The chemical composition of capsule varies according to organism but usually consist of CAPSULE STAINING polysaccharides e.g., glucose, galactose, amino sugars. Capsules are colorless and have low refractive index and so are difficult to observe without a special staining technique. Techniques like negative staining can be used to demonstrate the capsule. 71 Congo red: Principle Capsule staining is diagnostically useful since it is a virulent factor(e.g. pneumococci). Bacterial capsules are non-ionic, so neither acidic Congo red is a negative stain. It stains the background leaving the capsule unstained. nor basic stains will adhere to their surfaces. Acid fuchsin fixes congo red and reacts with it to give a Capsules are demonstrated either by negative blue background. staining (Nigrosin or India ink) or by special staining, e.g. Hiss’method, Anthony’s method Acid fuchsin also stains the organism pink, leaving the capsule colorless. 72  Observation SPORE STAINING The morphology of bacterial endospores is best observed in unstained wet films under the phase contrast microscope, where they appear as large, refractile, oval or spherical bodies within a bacterial mother cells or else from the bacteria. If spore-bearing organisms are stained with ordinary dyes , or by Gram’s stain , the body of the bacillus is deeply colored , whereas the spore is unstained and appears as clear area in the organism. This is the way in which spores are most commonly observed. 73 75 Spore staining If desired , however , it is possible by vigorous staining procedures to introduce dye into the substance of the spore. ENDOSPORE STAINING When thus stained , the spores tends to retain the dye after treatment with decolorizing agents. 76 Endospore staining Principle Spores are normally impervious to stains. An aqueous primary stain, malachite green is Under the light microscope endospores have a high light applied and steamed to enhance the penetration of refractivity indicative of high protein content. the impermeable spore coat. Endospores can be stained by: Once stained the endospore does not readily decolorize even with the application of decolorizer and Modified Zeihl-Nelson's method using 0.25-0.5% sulphuric acid they appear, but the cytoplasm of the vegetative cell as decolorizing agent, takes the color of safranine and appears red. Barthelomew-Mittwar’s method Schaeffer-Fulton stain technique. 79 Schaeffer-Fulton method Observation Malachite green is used to stain the endospores (primary stain) The malachite green is forced to permeate the spore wall by heating (mordant). Washing with water remove stain from vegetative cells, but not from spore wall. The endospores thus retain the primary dye while the vegetative cells lose the primary stain and take the red color of secondary stain (safranin). 80  FLAGELLAR STAINING Flagellar staining provides taxonomically valuable information about the presence and distribution pattern of flagella on prokaryotic cells. Bacterial and archaeal flagella are fine, threadlike organelles of locomotion that are so slender (about 10 to 30 nm in diameter) they can only be seen directly using electron microscope. To observe bacterial flagella with the light microscope, their thickness is increased by coating them with mordants such as tannic acid and potassium alum, and then staining with pararosalineor basic fuchsin. 81 Bacterial Flagella staining 82