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Practical Medical Microbiology Dr. Chandra Prakash Bhatt, PhD A.K. Books and Educational Enterprises This book is dedicated to MY LATE FATHER DEV. Good microbiological laboratory practice (GMLP). 3 Basic Pract Book 2/ 11/06 am Page 1 . Suitability for level of practical microbiological work. for authors of textbooks and laboratory manuals, and for instructors, is to project manual seeks to provide practical insight and experience rather than to detail.

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practical diagnostic medical microbiology Medical microbiology is the science which deals with the study of The book has been accordingly Essentials of M. Practical Microbiology: based on the Hungarian practical notes This book is freely available for research and educational purposes. Reproduction in any XML to PDF by RenderX XEP XSL-FO F ormatter, visit us at lesforgesdessalles.infox. com/. PDF | On Feb 2, , Dr. C. P. BHATT and others published PRACTICAL MEDICAL Download full-text PDF .. BOOK Practical Medical Microbiology.

Areas generally considered part of microbiology that were not covered or covered only briefly in thefirst edition are now included with comprehensive introductory chapters. These organisms die rapidly on exposure to air. When more quantitative measures are required, a microbial risk assessment model may bethe method of choice. SchusterChapter 46 Introduction to Yeasts Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or uti-lized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopy-ing, microfilming, and recording, or in any information storage or retrieval system, without written permission from thepublishers. It is important to differentiate active motility from brownian movement. Antony Von Leeuwenhoek is considered to be the first person who has seen a micro organism through a simple microscope made by him with a magnification of times.

Staining 8 3. Gram staining method 11 4. Ziehl- neelsen staining method 17 5. Bacterial spore staining method 27 7. Potassium hydroxide KoH preparation 31 9. Lactophenol cotton blue stain 33 Thick and thin blood smear examination 35 Microscopic examination of stool 40 Safety in microbiology laboratory 50 Quality control in microbiology laboratory 53 Microbial control 55 Culture of bacteria and cultural characteristics 64 Culture media 68 Aerobic and Anaerobic culture 76 Blood culture 79 Urine culture 85 Cerebrospinal fluid culture 94 Sexually transmitted disease 98 Respiratory tract infection Mycobacterium culture Gastrointestinal tract infection Pus culture Eye infection Body fluids culture Pyrexia of unknown origin PUO Water borne disease Food borne disease Antibiotic sensitivity test Biochemical tests Serological tests used for diagnosis of microbial diseases Antistreptolysin O ASO test Complement fixation test C-reactive protein CRP test Dick test Elek test Lepromin test Quellung test Rheumatoid factor RF test Schick test Serological diagnosis of human immuno deficiency virus Serological diagnosis of syphilis Decolourisation is a critical procedure.

Streptococcus pneumoniae In pairs and short chains: From liquid culture 1 Take clean. Staphylococcus aureus In chains: Enterococcus species Gram positive bacilli In chains eg. Escherichia coli Klebsiella pneumoniae Comma shaped. The appearance of purple coloured Gram positive bacteria and pink coloured Gram negative bacteria in the control smears indicate proper staining technique and stained test smear is compared with it.

Neisseria gonorrhoea Neisseria meningitidis Gram negative bacilli eg. Wipe the slide with a cotton swab dipped in alcohol and then holding its end with forceps roast it free from grease by passing 6 — 12 times through a blue Bunsen flame. Preparation of bacterial smear: From solid media 1 Take clean and grease free glass slides for making the smears. Hold the slide with hand on one corner in a slant position and add drop by drop of absolute alcohol till a faint colour comes out of the smear.

List of Gram positive and Gram negative bacteria are summarized in the table Streptococcus species Lanceolate shaped in pairs: Staphylococcus species and Gram negative bacilli eg. Bacillus anthracis with spores eg. Allow it to stand for one minute. Streptococcus species in exudates from tonsillitis. This modification is recommended for examination of smears for gonococci and meningococci.

Gram negative rods in pus from cases of chronic otitis media and post operative wound infections. Clostridium difficile. What are the other differential stains used for staining? The conditions that can result in Gram positive bacteria appearing Gram negative are: Describe differences between a Gram positive and Gram negative cell wall.

VIVA 1 Define differential stain. The important differences between Gram positive and Gram negative cell wall are as follows: Differential stain is defined as those that contain two different coloured dyes.

Other examples of decolourizing agents are acetone. Other examples of counterstains are basic fuchsin and neutral red. These changes allow crystal violet to come out of the cell wall during process of decolourizing resulting in some cells staining pink and others staining purple. It is dangerous to add the water to the acid. When stained they appear pink against a blue background. The flask containing solution is kept over a boiling water-bath for about 5 minutes.

In this staining method. The tubercle bacilli resist the decolourizing action of acid-alcohol which confers acid fastness to the bacteria. Great care must be taken to avoid spilling the acid on skin. The acid must be added to the water. The degree of acid fastness varies in different bacteria. Remainder of acid is added in same manner. PRINCIPLE Acid fastness of acid-fast bacilli is attributed to the presence of large quantities of unsaponifiable wax fraction called mycolic acid in their cell wall and also the intactness of the cell wall.

The ordinary aniline dye solutions do not readily penetrate the substance of the tubercle bacillus and therefore is unsuitable for staining. The Ziehl-Neelsen acid-fast staining method has proved to be most useful for staining acid fast bacilli belonging to the genus Mycobacterium especially Mycobacterium tuberculosis and Mycobacterium leprae. This is prepared by adding 95 ml of ethanol and adding water to it to make ml. The other microorganisms.

Then ml of distilled water is added to it and the mixture is filtered before use. The non-acid fast bacilli readily absorb the colour of the counter. These acid fast bacilli have a high concentration of the lipid-mycolic acid in their cell walls. Once stained. Ehrlich suggested the method for the differential staining of tubercle bacilli and other acid-fast bacilli with anilinegentian violet followed by strong nitric acid.

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The mixture becomes hot. Different modifications of acid-fast staining is summarized in the box Preparation of strong carbol fuchsin: This solution is prepared by dissolving 5 grams basic fuchsin powder in 25 grams crystalline phenol by placing them in a 1 litre flask. When the solution is complete. Findings are recorded. List of other acid—fast structures are provided in the table Then hydrochloric acid is added slowly and the top of the flask is covered to stop the fumes from escaping.

The smear should be examined following a zig-zag pattern for at least 10 minutes or fields. This solution contains 75 ml concentrated hydrochloric acid HCl and 25 ml of industrial methylated spirit.

The flask is placed in 5—8 cmm of cold water in the sink. It is left for 10 minutes. Do not allow the stain to dry on the slide. The smear is positive for acid fast bacilli. With appropriate staining.

Allow the smear to be air dried. It is then decanted into a labeled bottle for use. Allow it to stain for 5 minutes. Frequently examined specimens for the detection of Mycobacterium tuberculosis are summarized in the box Methylated spirit is poured into a large flask.

Bronchial or laryngeal washings. Renal tuberculosis Urine. Acid fast bacilli appear pink coloured in stained smears. Miliary tuberculosis Bone marrow. Liver biopsy. Tuberculous meningitis Cerebrospinal fluid. Gastric lavage when sputum is swallowed as in children. At the end of the process of decolourisation by sulphuric acid. Acid fastness of the bacteria is attributed to presence of mycolic acid in high concentration in the cell walls of tubercle bacilli and also to the intactness of the cell wall.

What are the various specimens obtained in the laboratory for the diagnosis of tuberculosis? Some bacteria are acid-fast because their thick waxy cell wall is made up of long chain fatty mycolic acids.

These mycolic acids render the cells resistant to decolourisation. Thus when acid is added as a decolouriser. List different modifications of acid fast staining and their uses. Why are some bacteria acid fast? List another method that can be used for detecting acid fast bacteria in a smear? By this method acid fast bacteria fluoresces bright yellow or orange against a greenish background.

Fluorescent staining methods using fluorochrome dyes such as auramine O and rhodamine is another method that can be used for detecting acid fast bacteria in a smear.

List the acid fast organisms. Toluidine blue stains the granules bluish black due to metachromatic effect and malachite green stains the bacilli green. The diphtheria bacillus.

Corynebacterium diphtheriae has well developed granules within their bacterial cytoplasm. With basic dyes. This stain is composed of 1.

These granules are known as Volutin granules. Toluidine blue and malachite green are dissolved in the alcohol and then added to the water and acetic acid. Allow it to act for 1 minute. The solution is made by first dissolving 2 gram potassium iodide in 10 ml distilled water and then 1 gram of iodine is further added to it with dissolution.

These granules are made up of polymetaphosphate and are seen in unstained wet preparations as round. Babes Ernst granules.

Allow the smears to be air dried. II Reagents and glass wares Bunsen flame. With toluidine blue or methylene blue. The stain is then allowed to stand for one day and then filtered. Allow it to stain for minutes.

VIVA 1. What is the reason for such an arrangement? The typical arrangement of C. The method is highly specific as well as sensitive. The significance of metachromatic granules is that they represent storage depots of materials needed to form highenergy phosphate bonds.

When a substance is stained with a particular coloured dye. What is metachromasia? This method involves the use of specific antibodies raised against C. This phenomenon is observed with C. The smear is positive for bacilli showing bluish-black metachromatic granules. They are not found during active growth period and are depleted under starvation conditions. Metachromatic granules are composed of polymetaphosphate. This is due to incomplete separation of the daughter cells after longitudinal binary fission.

The granules present in the bacteria are called metachromatic granules because the blue colour of the stain is changed to bluish-black by those granules. Their presence in thin slender bacilli helps to distinguish C. The smear is viewed by immunofluorescent microscopy. They are the positive and negative staining procedures. When the capsule has a diameter of 20 nanometer or more and seen under light microscope.

Capsule staining is more difficult than other types of differential staining procedures because. When the diameter is less than 20 nm and seen under electron microscope. In the positive staining technique. Two types of staining procedures can be employed to demonstrate the capsule. Dry-film negative staining methods using India ink.

Nigrosin staining is prepared by adding 0. Two dyes. The best method for staining capsules on bacteria in either liquid or solid media is the wet-film India ink method. Capsules may be seen in stained or unstained preparations as a clear zone around the bacteria. The smear is not heat fixed. The capsule is non-ionic. Bacterial smears should not be heated. Negative staining method: The wet India-ink film contains capsulated bacteria.

Textbook of Practical Microbiology 35 For negative staining of smears 1 Take a clean grease free glass slide. Observation of negative staining method The capsule in negative staining method is seen as clear refractile.

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Bacillus anthracis in the stained blood smears is demonstrated by McFadyean reaction which uses polychrome methylene blue. In negative staining. The smear shows capsulated bacteria. Neisseria memigitidis fresh isolates capsulated Bacillus anthracis polypeptide capsule Clostridium perfringens Bacteriodes fragilis Escherichia coli some strains Klebsiella pneumoniae Vibrio parahaemolyticus Yersinia pestis Francisella tularensis Haemophilus influenzae Bordetella pertusis 3 Describe the functions of the capsule.

Group B streptococci Group D streptococci polysacchaide capsules Streptococcus pneumoniae. Red coloured-safranine as counterstain is used as the second reagent to colour the decolourised vegetative cells. The application of heat facilitates penetration of the primary stain. The spore remains green. Preparation of safranine stain: This stain is prepared by dissolving 0.

Spore production is a very important characteristic of some bacteria such as members of anaerobic genera Clostridium and aerobic genus Bacillus. These are malachite green stain Schaeffer and Fulton method. Preparation of malachite green stain: This stain is prepared by dissolving 5 gram of malachite green in ml of distilled water.

On the other hand. The spores on staining with modified Ziehl-Neelsen stain appear red and bacilli blue. Different staining techniques are available for staining of spores. Unlike most of the vegetative cells that are stained by common procedures. After the primary stain is applied and the smear is heated. They occur when environmental conditions become unfavorable for continuing vegetative cellular activities. They are highly resistant and metabolically inactive forms.

The spores retain the green of the primary stain. II Reagents and lab wares Bunsen burner. Ordinary tap water acts as decolourising agent. Because of the chemical composition of spore layers. Once the spore is stained with the malachite green. The morphology of the bacterial endospores is best observed in unstained wet films under the phase-contrast microscope.

The spores of non toxigenic strain of Clostridium tetani are used for testing efficacy of sterilization by dry heat. Spore bearing bacilli causing anthrax is Bacillus anthracis. Allow it to act for 30 seconds. Clostridium septicum. It represents red coloured vegetative bacilli with green coloured spores by the malachite green staining method. The spores of Bacillus stearothermophilus are used for testing efficacy of sterilization by moist heat.

May be sporebearing bacilli eg. On a clean glass slide. Spore bearing bacilli causing gas gangrene are Clostridium perfringens.

Clostridium novyi. Record the observations in the note book. VIVA 1 Name spore bearing bacilli causing anthrax and gas gangrene. Instead it forms a deposit around the organism. The procedure requires the use of acidic stains such as India ink or Nigrosin. In this chapter dry film negative staining by using nigrosin to detect bacteria in dry smears will be described.

Wet film India-ink method is the best method for staining capsules of bacteria from cultures in either liquid or solid media. Negative staining finds its utility for the demonstration of capsule and bacteria difficult to stain such as Treponema palladium. II Reagents and glass wares These include Bunsen flame. Cryptococcus neoformans. Indian ink method for demonstration of capsule is described earlier in the chapter Nigrosin staining solution is prepared by adding 0.

The slide is then pushed away from the previously spread drop of suspended organism. Negative staining procedure is so called because the background gets stained and the organism remains colourless. The different stains employed for negative staining are: India ink. This is particularly useful in the demonstration of bacterial capsules. Which bacteria can be demonstrated by India Ink. VIVA 1 List the different stains that can be employed for negative staining.

The choice of most appropriate media depends on many factors including nutritional and growth requirements of the bacteria. This medium is a selective medium for growth of Salmonella spp. These are: These contain nutrients that support the growth of non-fastidious bacteria. Selenite F broth. Some media may contain additional nutritional supplements. Since bacteria have varied growth requirements.

Media generally contain a carbon source. These media contain ingredients that selectively enable the growth of some species. This medium inhibits Escherichia coli and other Gramnegative bacteria. These are solid selective media.

These media differentiates between species of bacteria depending on a specific property. Deoxycholate citrate agar DCA medium. They do not confer any selective advantage. They favor the growth of some bacteria by extending the lag phase of others eg.

Meat extract and peptone are the commonest sources of carbohydrates and amino acids. MacConkey Fig. In addition solid media contain agar as a solidifying agent.

These are liquid selective media. These media. Basal medium Differential medium Selective medium Enriched. Jensen medium Fig. Media are of different types. This medium is used to demonstrate lactose fermenting properties.

Colonies of non-fastidious bacteria such as S. II Reagents and media Different kinds of media such as nutrient agar. Haemolytic strain of S. MacConkey agar and Selenite F broth. Proteus mirabilis and Salmonella spp. In addition S. III Specimen 24 hour broth cultures of Staphylococcus aureus.

Growth positive Salmonella spp. Ans Enriched medium e. Geneva Guidelines on standard operating procedures for Microbiology. Selenite F broth inhibits all except S. Proteus spp. World Health Organisation. Chapter 6: Cultivation of bacteria on laboratory media. VIVA 1 Give examples of different kinds of media used for culture of bacteria. Salmonella spp. SOP 54 Version: In SF broth most bacteria are inhibited.

Microbiological Methods. Inoculation of Culture Media. American Society for Microbiology. Vijayalakshmi N. Pseudomonas S. Uncultivable bacteria: Implications and recent trends towards identification.

Ed Clinical Microbiology Procedures Handbook. Indian J Med Microbiol. Washington DC. Streptococcus spp. Parija SC. Most of the bacteria grow on enriched media. Blood safety and clinical technology. Pseudomonas spp.

II Reagents and media Nutrient agar plates. Apart from nutritional requirements. Examples of bacteria showing different temperatures for their growth is summarized in the table Either event causes biochemical reaction in bacterial cells to cease. Above this point. Based on their temperature requirements. A number of factors influence the growth and culture of bacteria on culture media. In general. These factors include availability of proper temperature.

The ideal temperature for growth may not coincide with that for specific enzyme activities. Table Examples of bacteria showing different temperatures for their growth Type of bacteria Psychrophilic Mesophilic Thermophilic Example Psychrobacter immobilis. Different bacteria require different optimal temperature for their growth. VIVA 1 How does temperature affect bacterial growth? Bacterial growth is dependent on enzyme activity. Methanosarcina spp. At high temperatures.

There is a different temperature requirement for optimal growth and pigment production. Pigment production will be seen with the cultures of both Flavobacterium and S.

Enzyme activity in turn is influenced by temperature. Thermus aquaticus. Each species has an optimum range of temperature within which its reproductive rate and hence growth is optimum. Bacillus stearothermophilus. Flavobacterium spp is capable of growth at low temperature as well. Pigment production. II Reagents Nutrient broth tubes. Most pathogens show an optimum pH close to that of their preferred habitat.

Alcaligenes faecalis and Candida spp. This is a reflection of this natural environment eg. Most laboratory media have a neutral pH which suits nearly all organisms. Salts of weak acids and weak bases may also be added. The ability to grow at different pH varies among bacteria. Like temperature. These may include natural buffers like proteins. Each species of bacteria can grow within a particular pH range. Vibrio cholerae and Enterococcus faecalis. Most body fluids have a pH of 7.

Pathogenic bacteria have adapted to their hosts. Citrate buffer. VIVA 1 Name some buffers used in preparing culture media. Facultative anaerobes are evenly dispersed throughout the medium while microaerophiles grow slightly below the surface subsurface growth. Pseudomonas aeruginosa. These bacteria can grow in the presence or absence of free oxygen. Oxygen requirement vary widely among bacteria and this is reflective of the different bio-oxidative enzyme systems present in bacterial cells.

They generally follow an aerobic respiratory pathway. These bacteria can grow only in the presence of free oxygen. When inoculated into a tube of semisolid or liquid medium. These bacteria require small amount of oxygen for their growth and survival.

An excess of oxygen is inhibitory to their growth. These bacteria cannot survive in the presence of free oxygen. They do not use oxygen as a final electron acceptor but possess enzymes like superoxide dismutase and catalase. Oxygen is one of the most important growth limiting factor for microorganism. It plays a vital role in many biological processes of the microorganisms..

Enzyme systems present in these bacteria require oxygen to be the final electron hydrogen acceptor especially for the oxidative breakdown of high energy molecule like glucose.

Aerobes will grow on the surface of the medium. The benefits are because of their ability to act as a final electron acceptor in the respiratory pathway with the liberation of much energy. However it also results in the accumulation of toxic byproducts. VIVA 1 Give example of obligate aerobes. Deep nutrient agar tubes are the simplest method.

Bacteroides spp. Strict anaerobes will not grow within a centimeter of the surface. II Reagents and media Blood agar plates and thioglycollate broth culture. In many laboratories. Anaerobic jars are a constant feature of anaerobic culture.

Textbook of Practical Microbiology

They include the McIntosh and Fildes jar Fig. Anaerobic condition should be checked by alkaline methylene blue indicator. Cooked meat particles also act as a good reducing agent Example. The resulting reaction liberates hydrogen and carbon dioxide. McIntosh and Fildes jar and Gaspak system. Anaerobes grow in the depths of the medium.

Water is added to the sachet and it is immediately placed in the jar. These organisms die rapidly on exposure to air. List of anaerobic bacilli and cocci are summarized in the box An indicator is also added to demonstrate anaerobiosis. The tubes are inoculated while still molten. A simpler but more expensive technique is the Gaspak system.

Obligate anaerobes can grow in media only in the absence of oxygen. For culture of anaerobes. The catalyst. Robertson Cooked meat medium Fig. What is the Hungate procedure of anaerobiosis? In the presence of an oxygen free gas. This ensures anaerobic conditions inside the tube. The organisms are then inoculated. The Hungate procedure is a method for the anaerobic culture of bacteria. The tubes are then sealed. These include a earthenware candles e. Moist heat: Moist heat kills the microorganisms by coagulating their proteins and denaturing their enzymes.

The hot air oven is commonly used in a microbiology laboratory for sterilization of laboratory glassware. The common methods used for sterilization of media is heat and filtration.

In a hot air oven. II Reagents Sterile nutrient broth. Pasteurisation is an example of sterilization by moist heat Box Seitz filter Fig. Different methods of sterilization of various substances are summarized in the table This is vital for isolation and maintenance of microbes.

Moist heat has greater penetrating power than the dry heat and so relatively lower temperature is required for sterilization by this method. Dry heat: Sterilisation by dry heat kills the bacteria by oxidizing essential cell components of the cell.

Sterilization by filtration Heat labile fluids such as serum.

I Equipments Autoclave Fig. There are different types of filters. Of these cellulose membrane filter is most extensively used now a days. The other two tubes will remain free of growth. Efficient filters should be able to retain Serratia marcescens.

If the correct sterilization conditions have been achieved no growth should occur. Color change from red to green is a satisfactory result. Filter paper strips impregnated with spores of B. No growth should occur. However moist heat is more effective requiring less time and temperature. Both dry and moist heats are effective sterilization methods.

Filtration is also a suitable method of sterilization. Sugar solution. VIVA 1 Why should media not be sterilized in large quantities? Large quantities of media are difficult to sterilize by heat. It is sufficient to kill heat labile bacteria. Heat stable media such as nutrient agar. Two methods are available: Biohazardous material. Some pathogens such as Coxiella burnetii is not destroyed.

These are sterilization by red heat. If methods like filtration are used. These include time. It will not be possible to ensure that all parts of the media have attained sterilization temperature. Heat labile media. The process reduces microorganism to a level acceptable for a defined purpose. The article to be disinfected should be cleaned thoroughly to reduce organic matter which may inactivate the agent or immersed in the disinfection for the required amount of time.

Commonly used disinfectants and their mechanism of actions are summarized in the box The fresh disinfectant will be able to bring about disinfection more effectively than the used one. II Reagents and media Sterile nutrient broth. With time and use. They have markedly different activity against different microorganisms and are generally most effective against Gram positive bacteria.

This will show a growth on the nutrient agar plate. Table summarises the list of commonly used disinfectants and antiseptics. The concentration at which they are used should be accurate for optimal activity. A disinfectant that can be safely applied to living tissue is called an antiseptic. A chemical agent that disinfects a substance is called a disinfectant. It acts by oxidation. They act by denaturing proteins damaging lipid complexes and dehydration.

Chlorine is a widely used disinfectant. They act on the cell membrane of bacteria. Iodine is commonly used as an antiseptic. They can also be used as antiseptics and are active against viruses as well.

Phenol is the standard disinfectant against which other disinfectants are compared. Antiseptics can be used for decontamination of living tissue. Disinfectants are used for decontamination of non-living tissue. They merely ensure that an object is relatively free of microbial contamination.

It is essential to test disinfectant from time to time to detect loss of activity. VIVA 1 What are the commonly used disinfectants and antiseptics?

Sterilization frees an article of all infectious materials including spores. They act by inactivating cellular protein.

The compounds act by the liberation of nascent oxygen and are effective against most bacteria and viruses.

They help in mechanical removal of microorganism. They act by a number of mechanisms such as disruption of cells. Catalase degrades the hydrogen peroxide in the bacterial cell before it can do any damage to the bacterial cell.

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Catalase is an enzyme produced by many bacteria. If stored in colourless penicillin vials. List of catalase positive and negative bacteria are summarized in the table Catalase test is also carried out for Mycobacteria to differentiate tubercle bacilli from atypical mycobacteria Box The enzyme splits hydrogen peroxide into water and oxygen. The catalase test is most commonly used to differentiate members of the family Micrococcaceae from members of the family Streptococcaceae Box The enzyme converts hydrogen peroxide into water and oxygen.

Hydrogen peroxide is a by product of aerobic respiration and is lethal if it accumulates in the bacterial cell. Muller-Hinton agar is tested. If catalase is absent. Tube method Production of the enzyme catalase can be demonstrated by adding hydrogen peroxide to colonies of the bacteria.

Use of this solution will give false negative results. Hydrogen peroxide must be stored in amber coloured bottles. If catalase is present it is indicated by the presence of free gas bubbles.

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II Specimen Pure growth of bacteria from solid media preferably from nonblood agar plates Examples: Effervescence indicates catalase production. Tube method Gas bubbles are released when colonies are introduced into the hydrogen peroxide in the test tube.

It is allowed to stand for a few minutes. Most strains of mycobacteria. Most atypical mycobacteria are strongly catalase positive. Catalase production is assessed by a Relative activity of the enzyme catalase determined by the height of the column of oxygen bubbles formed by the action of untreated enzyme produced by the organism Semi quantitative catalase test. It means bacteria possesses the enzyme catalase. On the basis of the semi quantitative catalase test. Staphylococcus aureus catalase positive bacteria.

Negative control: Streptococcus species catalase negative bacteria. Name some catalase positive and negative organisms. High concentration of H2 02 is toxic to the cell. Most of the aerobes are catalase positive. Most of the anaerobes do not possess the enzyme. University of Texas. VIVA 1 2 3 4 5 What is catalase test? What are the positive and negative controls used in the catalase test?

What is the importance of catalase test? Can you take colonies from blood agar plate for testing catalase reaction? Give explanations. In aerobic organisms. Care must be taken while performing catalase test in growth from blood agar plate. Houston Medical School.

Blood RBC contains catalase. Oxidase reagent is freshly prepared in distilled water every day. Direct plate technique 1 Take a nutrient agar plate with colonies of bacteria to be tested. This enzyme oxidises the reagent N-N tetramethyl para-phenylene diamine hydrochloride a colour less reagent in reduced form to indophenol blue. Tetramethyl — p-phenylene diamine. PRINCIPLE The cytochromes are iron containing haemoproteins that act as the last link in the chains of aerobic respiration by transferring electrons hydrogen to oxygen.

List of oxidase positive and negative bacteria is presented in the table Indirect filter paper strip procedure 1 Take a filter paper strip. The enzyme oxidase plays a vital role in the operation of the electron transport system during aerobic respiration. II Specimen Young culture of bacteria to be tested.

Cytochrome oxidase catalyzes the oxidation of a reduced cytochrome by molecular oxygen. Aerobic bacteria.

The cytochrome oxidase test uses certain reagent dyes such as p-phenylene diamine dihydrochloride which acts as a substitute for oxygen as artificial electron acceptors. Camphylobacter spp. All genera in family Enterobacteriaceae 2.

Iron wire loops should not be used. Fresh reagent must be used. Escherichia coli oxidase negative bacteria. Nichrome wire loops should not be used.

Flavobacterium spp. Results must be observed within 10 seconds. Francisella tularensis 6. Pseudomonas aeruginosa oxidase positive bacteria. Vibrio spp. Haemophilus spp. Indirect filter paper strip procedure In a positive test. Pasteurella multocida Gram negative cocci 1. In filter paper test. Achromobacter spp. Acinetobacter calcoaceticus 3. Table List of oxidase positive and negative bacteria Oxidase positive bacteria Gram negative rods 1. Bordetella spp except B.

Alcaligenes spp. In a negative test the colour of the colonies remain unchanged. Brucella spp except B. Colonies should not be picked up from selective media.

In a negative test the colour of the smear in the zone of the filter paper remain unchanged. Eikinella spp. Pseudomonas spp except Ps.

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Plesiomonas spp. Moraxella spp. Neisseria spp. Bordetella parapertusis 4. Chromobacterium spp. Cardiobacterium spp. It means bacteria possesses the enzyme oxidase. Brucella canis 5. Aeromonas spp. Branhamella spp. Oxidative negative bacteria 1.

Tetramethyl derivative of p-phenylene diamine is recommended because the reagent is i more stable in storage. Colour change must be noted within 10 seconds. In reduced state the dye is colourless and in oxidized state deep purple in colour.

Muller-Hinton agar.