Cultivation of Microorganisms

Cultivation of Microorganisms

CULTIVATION OF MICROORGANISMS OBJECTIVES:  To know the proper technique of cultivating microorganisms  To determine the factors that affect the growth of microbes  To familiarize oneself with the cultural characteristics of some microorganisms INTRODUCTION The survival of microorganisms in the laboratory, as well as in nature, depends on their ability to grow under certain chemical and physical conditions. An understanding of these conditions enables us to characterize isolates and differentiate between different types of bacteria. Such knowledge can also be applied to control the growth of microorganisms in practical situations. Methods of Culturing Microorganisms 5 Basic Techniques: - to manipulate, grow, examine and characterize microorganisms 1. inoculation - to culture, one introduces a tiny sample (inoculum) into a container of nutrient medium, which provides an environment in which they multiply 2. incubation 3. isolation 4. inspection 5. identification Culture – a culture is the microorganisms that grow in a culture medium – the observable growth that appears in or on the medium Colony – a pile or mass of a sufficiently large number of cells, growing on or in solid medium, that they are visible to the naked eye Types of Culture 1. Pure Culture – a container of medium that grows only a single known species or type of microorganism – most frequently used for laboratory study because it allows the systematic examination and control of one microorganism by itself 2. Mixed Culture – a container that holds two or more identified, easily differentiated species of microorganisms

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3. Contaminated Culture – was once pure or mixed but has since had contaminants introduced into it like weeds into a garden. Techniques for isolating cells The isolation methods most commonly used to get a pure culture include: streak plates and pour plates. •

Streak Plate Method – a small droplet of culture or sample is spread over the surface of the medium according to a pattern that gradually thins out the sample and separates the cells spatially over several sections of the plate o most commonly used isolation technique in microbiological laboratories o involves techniques for physically separating microorganisms on an agar surface o Streaking is a method of applying cultures to solid medium:  a sterile loop is cooled and brought into contact with a culture  the loop is then brought into contact with the surface of solid medium whereupon it is streaked (i.e., dragged) along the surface of the solid medium  colonies grow along the points of the streak o Colony isolation:  A petri dish is streaked in manner such that individual colonies may be isolated.

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•

Loop dilution or Pour Plate – the sample is inoculated serially into a series of cooled but still liquid agar tubes so as to dilute the number of cells in each successive tube in the series. o involves diluting organisms in fluid and adding a dilution to melted agar, and pouring agar into plate

Culture medium [pl. media] – Culture media are solutions containing all of the nutrients and necessary physical growth parameters necessary for microbial growth. – nutrient material prepared for the growth of microorganisms Criteria for culture medium: 1. contain nutrients for growth 2. proper moisture, oxygen and pH 3. sterile (initially) 4. incubated proper temperature –

Medium is always the singular form of the word, and media is always and only the plural form. This can be classified on three primary levels: (1)physical form (2)chemical characteristics (3)functional type

PHYSICAL STATE 1. Liquid Media − water-based solutions that do not solidify at temperatures above freezing and that tend to flow freely when the container is tilted 2. Semi-solid Media − exhibit a clot-like consistency; contains an amount of solidifying agent (agar or gelatin) that thickens them but does not produce firm substrate. − used to determine the motility of bacteria and to localize a reaction at a specific site 3. Solid Media − provide a firm surface on which cells can form discrete colonies − advantageous for isolating and subculturing bacteria and fungi – has physical structure (broth lacks structure) and this allows bacteria to grow in physically informative or useful ways (e.g., as colonies or in streaks). – usually used as:  slants 3


 

stabs petri dishes

a. Liquefiable Solid Media − sometimes called reversible solid media − contain a solidifying agent that is thermoplastic – its physical properties change in response to temperature Agar – complex polysaccharide isolated from the red algae (Gelidium) – the major solidifying agent used in bacteriological media – dissolves at approximately 100°C, and an agar-containing medium thus heated will not solidify until the temperature is brought down to about 43°C. Once solidified, the medium will not melt until brought back up to about 100°C – among the advantages of this interesting temperature-related property are the following:  medium can be inoculated while in a liquid state at a low enough temperature (approx. 43-50°C) such that the cells will not die off  the medium, once solidified, will stay solid over a wide range of incubation conditions – resistance to degradation by nearly all organisms and its relative clarity, permitting easy viewing of growth on or in the medium – very difficult, if not impossible, to purify it fully of trace impurities. Thus, when agar is added to a chemically-defined liquid medium, the medium must be considered complex. If an absolutely chemicallydefined solid medium is required, silicon-based solidifying agents can be employed. – In 1881, Fanny Eilshemius Hesse, a technician in the laboratory of Robert Koch in Germany, introduced the concept of agar to bacteriology, having used it for many years in the preparation of homemade jellies. – Short History:  Agar, as a culture medium, was suggested by Frau Hesse, the wife of a co-worker of Dr. Robert Koch. Walther Hesse apparently discussed his work with his wife, who was not a scientist, but did know her way around the kitchen. Lina Hesse - originally Fanny Angelina Eilshemius from NY - suggested that they try agar-agar when asked why her jellies and puddings stayed solid in the hot summers in Dresden. Walther told Koch about it. Dr. Robert Koch used it in 1881 to culture bacteria. It has been used ever since. Important properties: – few microbes can degrade it; remains solid even when bacteria and fungi are growing on them

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– –

melts at boiling point of water; does not melt below 100oC; can be used to culture many thermophiles once solidified can be incubated at temperatures approaching 100oC b. Nonliquefiable Solid Media − less versatile applications than agar media because they are not thermoplastic

CHEMICAL CONTENT OF MEDIA 1. Defined/Synthetic Media − media whose compositions are chemically defined − contain highly pure organic and inorganic compounds that vary little from one source from another − have a molecular content specified by means of an exact formula − all the ingredients of a culture medium are known, both qualitatively and quantitatively − great value in studying the nutritional requirements of microorganisms or in studying a great variety of their metabolic activities 2. Complex/Nonsynthetic Media − contain at least one ingredient that is not chemically definable − not a simple, pure compound and not representable by an exact chemical formula − exact chemical composition is not known, and such a medium is often prepared from very complex materials − contains most of the organic compounds-sugars, amino acids, and nucleotides-necessary for growth − contains nutrients released by the partial digestion of yeast, beef, soy or proteins − e.g. blood, serum, and meat extracts or infusions, body fluids, tissue extracts and infusions, and peptones, nutrient broth, Tryticase soy agar, MacConkey agar 3. Enrichment media - media used to enhance the growth of the desired organism in a mixed population; similar to selective media but designed to increase the numbers of desired microorganisms to a detectable level without stimulating the rest of the bacterial population Peptone – is a commercially-available digest of a particular plant or animal protein, made available to organisms as peptides and amino acids to help satisfy requirements for nitrogen, sulfur, carbon and energy – short chains of amino acids produced by enzymatic digestion of proteins

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SELECTIVE AND DIFFERENTIAL MEDIA 1. Selective Medium – contain one or more agents that inhibits the growth of a certain microbe or microbes (A, B, C) but not others (D), and thereby encourages or selects microbe D and allows it to grow – very important in primary isolation of a specific type of microorganisms from sample containing a highly mixed population – growth media that contains substances that inhibit the growth of unwanted organisms but permit the growth of the desired organism - uses certain dyes, high salt concentration, pH or antibiotics – Examples of selective media include:  mannitol salts agar (selects against non-skin flora)  MacConkey agar (selects against gram-positives)  eosin-methylene blue agar (selects against gram-positives)  phenylehyl alcohol agar (selects against gram-negatives)  Eosin, methylene blue, crystal violet dyes (inhibit the growth of gram-positive bacteria without affecting gram-negative) – supports the growth of desired organisms while inhibiting the growth of many or most of the unwanted ones – either by purposely adding one or more selective agents which "poison" certain types of organisms or by including or deleting certain nutrients such that the desired organisms and few others are able to grow 2. Differential Medium – can grow several types of microorganisms but it is designed to highlight differences among these microorganisms – allow the growth of more than one microorganism of interest but with morphologically distinguishable colonies. – contains a combination of nutrient and pH indicators to visually differentiate bacteria that grow on or in it – frequently a solid medium on which colonies of a particular species will have a distinctive color or cause a change in the color of the medium. – Examples of differential media include:  mannitol salts agar (mannitol fermentation = yellow)  blood agar (various kinds of hemolysis)  MacConkey agar (lactose fermentation = yellow)  eosin-methylene blue agar (various kinds of differentiation) – allows two or more different organisms to grow, but it contains dyes and/or other components upon which different organisms act in various ways to produce a variety of end products or effects, often detected by variations in color. These differences are often very apparent among colonies of a mixed culture growing in a petri dish. Pure cultures, growing in separate tubes of the same differential medium, may also be characterized and differentiated from one another according to a particular biochemical characteristic. 6


CULTURAL CHARACTERISTICS OF THE COLONIES IN PLATES: Plate

Color

Form

Margin

Elevation

A B C (1) (2) (3) A. TSI Reaction: Butt – Slant – Gas Production – Possible Identity of the Sample: COLONY MORPHOLOGY Differentiating colonies: – Colony morphology gives important clues as to the identity of their constituent microorganisms. – Important classes of characteristics include:  size  type of margin  colony elevation  colony texture  colony pigmentation Colony size – Colony size is dependent not just on the type of organism but also on the growth medium and the number of colonies present on a plate (that is, colonies tend to be smaller when greater than ascertain amount are present) and on culture medium characteristics. Usually stabilizes after few days: – Colony size usually stabilizes after a day or two of incubation. – Exceptions include:  slow growing microorganisms  during growth under conditions that promote slow growth  With slow growth colonies may continue to experience growth past this time, especially if an effort is made to prevent solid medium from drying out. Type of margin – Colonies can vary in the shape of their margins.

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Illustration, variation in colony form

Colony elevation – Colonies can vary in their elevations both between microorganisms and growth conditions, and within individual colonies themselves. Illustration, variations in colony elevation

Colony texture Surface appearance: – Colonies can vary in their texture. – Possible textures include:  shiny to dull  smooth to wrinkled  rough  granular  mucoid  A shiny, smooth, and/or mucoid appearance tends to be associated with the presence of capsular material. Colony pigmentation – Colonies can come in a rainbow of colors.

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Examples of various colony morphologies. The appearance of colonies on a plate is species specific and can be very helpful in identifying isolates. NUTRITIONAL AND ENVIRONMENTAL FACTORS INFLUENCING MICROBIAL GROWTH Growth requirements - two categories: o Environmental - temperature, pH, osmotic pressure, oxygen o Chemical - water, sources of carbon and nitrogen, minerals, and organic growth factors Chemical Requirements Microorganisms

-

Nutritional

Classification

of

Organisms can be divided into groups based on their energy and carbon sources. Regarding the source of energy which becomes trapped in

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an organism's ATP, the various life forms may be categorized as either chemotrophs or phototrophs. • Chemotrophs obtain their energy purely from the oxidation of chemical compounds. • Phototrophs use light as the ultimate source of energy. Phototrophs include plants, algae, cyanobacteria, and the purple and green anoxygenic bacteria. As carbon is a major and essential element in all living things, organisms may also be classified according to the nature of their source of carbon. Organisms which assimilate organic compounds for their carbon needs are termed heterotrophs. Those which utilize carbon dioxide are called autotrophs. Another method of classifying organisms nutritionally is by the source of reducing power utilized. All organisms need reducing power in the form of electrons for biosynthesis. Organisms that oxidize organic compounds are called organotrophs and those that oxidize inorganic compounds are called lithotrophs. Considering the various requirements for carbon and energy described above, nearly all living things can be placed in one of the following categories: •

•

• •

CHEMOHETEROTROPHS. As these organisms are generally organotrophic, they may also be called chemoorganotrophs. These organisms may use a variety of organic compounds as both carbon and energy sources. A common sugar so used is glucose. ATP is generated by either substrate-level or oxidative phosphorylation. These also require organic molecules such as glucose, amino acids and vitamins, which supply carbon and energy or are vital growth factors. CHEMOAUTOTROPHS. As these organisms are generally lithotrophic, they may also be called chemolithotrophs. ATP is usually generated by oxidative phosphorylation. PHOTOHETEROTROPHS. Green nonsulfur and purple nonsulfur bacteria PHOTOAUTOTROPHS. Plants, algae and cyanobacteria use water to reduce carbon dioxide, producing oxygen as a byproduct

Energy source  Phototroph - light  Chemotroph - chemical Carbon source  Heterotroph -- "feeder on others" organic

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

Autotroph - "self-feeder" carbon dioxide

* Most medically important microorganisms are chemoheterotrophs. Environmental Factors affecting Microbial Growth The physical-chemical factors of natural environment determine the rates of microbial growth and the nature and size of the indigenous population. 1.

Temperature – one of the most important factors affecting the growth rate of microbes – a profound effect on microorganisms  For many bacteria both extremely high and extremely low temperatures can be quite harmful, the former due to protein denaturation, the latter due to intracellular ice crystal formation upon freezing.  There neverthless exist microorganisms whose optimum growth temperatures might be considered extreme. – as the temperature rises, there is a minimum temperature, below which growth does not occur – as we rise above the minimum, rate of growth increases in accordance with the laws governing the effect of temperature on the chemical reactions that make up growth – reactions are mostly enzyme catalyzed – a point is reached the optimum temperature when there is also a very rapid increase the rate of inactivation of heat sensitive cell components, like enzymes, ribosomes, DNA, membranes etc. – above an optimum temperature, this heat denaturation will occur so rapidly that there is a corresponding rapid drop in the rate of growth to give a maximum temperature for growth for that particular microorganism – there is an increase in the growth rate due to increasing the rates of enzyme reactions; eventually a temperature becomes too high and microorganisms are damaged by enzyme denaturation, membrane disruption, and other phenomena – organisms can be divided into groups on the basis of their preferred temperature range  psychrophile --- optimum <15oC; can even grow at temperatures below O oC; die at temperatures much above 20 oC, live in surface of glaciers, snowfields, ice and cold water --- do not cause disease to humans because they cannot survive body temperature

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mesophile --- optimum 20-45oC; human pathogens are mesophiles o e.g. Thermoduric organisms – mesophiles that capable of short exposures to relatively high temperatures; organisms that survive following inadequate heating of foods and may thereby contribute to the spoilage of foods that have been heated (e.g., Pasteurization) to kill microorganisms  thermophile --- optimum 55-65oC; above 45 oC; found in compost heaps or hot springs --- do not cause disease because they freeze at body temperature  hyperthermophile --- optimum > 80oC; e.g. Pyrodictium; usually members of the Archae and are found growing near hydrothermal vents at great depths in the ocean Organisms exhibit distinct cardinal temperatures (minimal, maximal, and optimal growth temperatures)  minimum temperature - temperature below which no growth occurs  optimum temperature - maximum growth; highest growth rate  maximum temperature - temperature above which no growth occurs 

–

2.

pH – – – –

–

–

the negative logarithm of the hydrogen ion concentration organisms are sensitive to changes in acidity because hydrogen ions and hydroxyl ions interfere with hydrogen bonding within the molecules of proteins and nucleic acids Most microbes grow best at pH near neutrality, bacteria usually slightly on the alkaline side and algae and fungi on the acid side. some can grow at extreme values of low or high environmental pH. For example, a few bacteria that oxidize inorganic sulphur compounds to H2SO4 can grow at pH 0 (i.e. 1M H2SO4) other bacteria, as those causing human urinary, tract infections (that hydrolyze urea to produce excess of NH3 causing a rise in pH) grow at high pH of 11.0 Each species has a pH growth range and pH growth optimum  acidophiles - grow best between pH 0 and 5.5; organisms that grow best in acidic habitats e.g. chemoautotrophic bacteria – live in mines  neutrophiles - grow best between pH 5.5 and 8.0; pH range of most tissues and organs in the human body  alkalophiles - grow best between pH 8.5 and 11.5 13


e.g. Vibrio cholerae – grows best outside of the body in water at ph 9.0 – Microorganisms can usually adjust to changes in environmental pH by maintaining an internal pH that is near neutrality; some bacteria also synthesize protective proteins (acid shock proteins) in response to pH – When bacteria are cultured in the lab, they produce acids as part of their metabolism. The acids can interfere with bacterial growth. Buffers (chemicals) are added to growth media to maintain proper pH. – measure of the amount of acidic ions vs. basic ions Scale of 1-14 1. most grow at pH of 7 (neutral) 2. acidophilic (acid-loving) e.g. Helicobacter pylori – acid-tolerant bacterium; neutralizes stomach acid by secreting bicarbonate and urease, an enzyme that converts urea to ammonia, which is alkaline 3.

Oxygen concentration Aerobe – An aerobe is a microorganism that can utilize molecular oxygen as its final electron acceptor, i.e., as in cellular (aerobic) respiration. – produce enzymes catalase and superoxide dismutase that protect them from toxic forms of oxygen –

 Obligate aerobe No fermentation: i. An obligate aerobe is a microorganism that cannot live in the absence of molecular oxygen. ii. This basically means that they cannot obtain energy via fermentative processes. iii. More precisely, obligate aerobes are organisms that 1. have an electron transport system 2. are able to grow in the presence of atmospheric oxygen concentrations 3. can use O2 as a final electron acceptor 4. cannot ferment 14


–

Examples: i. Bacillus subtilis ii. Bdellovibrio spp. iii. Bordetella pertussis iv. Legionella spp. v. Mycobacterium leprae vi. Mycobacterium tuberculosis vii. Neisseria gonorrhoeae viii. Neisseria meningitidis ix. Pseudomonas spp.

 Obligate [strict] anaerobe a. O2 intolerant: i. Many anaerobes not only can't utilize molecular oxygen but are harmed by it as well. ii. One usage of the term obligate anaerobe is to describe only those microorganisms which are unable to grow (and, for that matter, even survive) in the presence of molecular oxygen. iii. The other, less strict usage of the term obligate anaerobe is simply to distinguish the term "anaerobe" from the term "facultative anaerobe." iv. Of bacterium, which are incapable of survival in the presence of O2 are included Clostridium botulinum and Clostridium tetani. –

Facultative anaerobes - organisms that have the ability to use oxygen when it is present but are able to continue to grow by using fermentation or anaerobic respiration when oxygen is not available though their metabolic efficiency is often reduced in the absence of oxygen e.g. Escherichia coli

–

Aerotolerant anaerobes – do not use aerobic metabolism but they tolerate oxygen by having some of the enzymes that detoxify oxygen’s poisonous forms e.g. lactobacilli that transform cucumbers into pickles; milk into cheese; Streptococcus pyrogenes

– Microaerophile a. Low O2 requirement and tolerance: i. Microaerophiles are microorganisms which are unable to grow when oxygen concentrations reach those found in air (20%) but nevertheless whose growth requires the presence of some oxygen (2% to 10%).

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Microaerophiles are damaged by the 21% concentration of oxygen in the atmosphere because they have limited tolerance due to sensitivity to toxic forms of oxygen, low levels of detoxifying enzymes or enzymes that don't work adequately. iii. Microaerophiles appear to grow best in the presence of a small amount of free oxygen. They grow below the surface of the medium in a culture tube at the level where oxygen availability matches their needs. b. Examples: i. Borrelia burgdorferi ii. Helicobacter pylori ii.

4.

Osmotic pressure – is the diffusion of water across a membrane from an area of higher water concentration (lower solute concentration) to lower water concentration (higher solute concentration) – is related to the concentration of dissolved molecules and ions in a solution – a cell can find itself in one of three environments: isotonic, hypertonic, or hypotonic. (The prefixes iso-, hyper-, and hyporefer to the solute concentration)  In an isotonic environment, both the water and solute concentration are the same inside and outside the cell and water goes into and out of the cell at an equal rate.  If the environment is hypertonic, the water concentration is greater inside the cell while the solute concentration is higher outside (the interior of the cell is hypotonic to the surrounding hypertonic environment). Water goes out of the cell.  In an environment that is hypotonic, the water concentration is greater outside the cell and the solute concentration is higher inside (the interior of the cell is hypertonic to the hypotonic surroundings). Water goes into the cell. – different relationships with O2 are due to several factors including inactivation of proteins and the effect of toxic O2 derivatives (superoxide radical, hydrogen peroxide, and hydroxyl radical), which oxidize and destroy cellular constituents; many microorganisms possess enzymes that protect against toxic O2 derivatives (superoxide dismutase and catalase) – most bacteria require an isotonic environment or a hypotonic environment for optimum growth. Organisms that can grow at relatively high salt concentration (up to 10%) are said to be osmotolerant. Those that require relatively high salt

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concentrations for growth, like some of the Archea that require sodium chloride concentrations of 20 % or higher halophiles. i. Obligate halophile – adapted to growth under high osmotic pressure ii. Facultative halophile – do not require high salt concentrations but they can tolerate them Plasmolysis osmotic loss of water resulting in shrinkage of the cell; growth of the cell is inhibited as cytoplasmic membrane pulls away from the cell wall. - Environments containing large concentrations of dissolved substances draw water out of cells, causing shrinkage of the cytoplasm volume interferes with growth and this is why highly osmotic environments prevent bacterial growth (e.g., brine, the high sugar concentrations in jellies and jams, salting of meats) - addition of salts (or other solutes) to solution results in an increase in osmotic pressure, used to preserve food (salted fish, honey, and sweetened condensed milk are preserved largely by this mechanism) - high salt or sugar concentrations draw water out of microbial cells and prevent growth

DIFFERENT CULTURE MEDIA 1. Sabourauds’ Dextrose Agar − was formulated by Sabouraud in 1892 − a solid medium used for isolation, cultivation and maintenance of pathogenic and non pathogenic yeasts and molds  recommended for the cultivation and growth of fungi, particularly those associated with skin infections − peptone medium supplemented with dextrose to support the growth of fungi, and is inhibitory to contaminating bacteria in clinical specimens. The acidic pH, however, also may inhibit some fungal species. Emmons modified the original formulation by adjusting the pH close to neutral to increase the recovery of fungi and by reducing the dextrose content from 40 to 20 g/L − pH is adjusted to approximately 5.6 in order to enhance the growth of fungi, especially dermatophytes, and to slightly inhibit bacterial growth in clinical specimens − peptones are sources of nitrogenous growth factors while dextrose provides an energy source for the growth of microorganisms. This formulation serves as an excellent basal medium to which antibiotics and other inhibitors may be added for the selective cultivation of various groups of microorganisms.

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2. Blood/Chocolate Agar – a nutrient medium which is used in culturing fastidious organisms such as Haemophilus species and Neisseria – comprised of sheep blood that provides the X and V factors necessary for Haemophilus growth – in addition to 5% blood, it contains a rich amino acid source but minimal carbohydrates (fermentation of these would produce acids that would readily lyse the red blood cells). – some pathogens secrete products that damage the membranes of host cells. Production of these hemolysins can be detected on blood agar. – heat-lysed red-blood cells in agar – HEMOLYSIS: Appearance of culture in blood agar.  alpha-HEMOLYSIS: Partial hemolysis, appearing green.  beta-HEMOLYSIS: Full hemolysis. A "halo" appears around the colony.  gamma-HEMOLYSIS: No hemolysis. Hemolysis on Blood Agar – used for the preliminary or confirmatory identification of many types of clinically important bacteria. While it is factored into the differential diagnosis of a specific infectious agent, hemolysis type is not specific enough to be a final diagnosis criterion. 

Alpha-hemolysis is a greenish discoloration of the blood agar surrounding a bacterial colony; it is a characteristic of Streptococcus pneumoniae. This is an incomplete lysis or "greening" of red blood cells



Beta-hemolysis indicates a zone of clearing in the blood agar in the area surrounding a bacterial colony. It is a characteristic of Streptococcus pyogenes as well as some strains of Staphylococcus aureus. This is a complete lysis (hemolysis) of red blood cells surrounding the colonies

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Gamma-hemolysis is actually a lack of hemolysis in the area surrounding a bacterial colony growing on blood agar. In fact, culture of bacteria on blood agar for the purpose of hemolysis classification is performed at 37oC in the presence of 5% CO2. This results in an overall brownish discoloration of the blood agar, from its original blood-red hue. An uninoculated blood agar plate (BAP) is shown on the left, above. Gamma-hemolysis would therefore describe bacterial growth that results in neither a greenish tinge to the discoloration (alpha-hemolysis) nor a clear zone that the observer "could read a newspaper through" (beta-hemolysis). This has no lysis of red blood cells. Staphylococcus epidermidis is gamma hemolytic. – Purpose:  a non-selective medium for the primary isolation of fastidious bacteria such as Neisseria meningitidis and Haemophilus spp.  recommended as a primary plating medium for spinal fluids, eye cultures, gonococcal cultures, and any other specimen which may contain fastidious organisms 3. Coliform Test a. Triple Sugar Iron Agar (TSI) – used to differentiate enterics based on the ability to reduce sulfur and ferment carbohydrates – a differential media used for detection of glucose, lactose, and sucrose fermentation and the production of gas and H2S. It contains the pH indicator phenol red. The media will turn yellow in an acid pH. This test is normally used for fermentative gram-negative rods. – Purpose: – used for the differentiation of Gram-negative enteric bacilli based on carbohydrate fermentation and the production of hydrogen sulfide – to determine the ability of an organism to attack a specific carbohydrate incorporated into a basal growth medium, with or without the production of gas, along with the determination of possible hydrogen sulphide (H2S) production – Principle: 

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contains three sugars (dextrose, lactose and sucrose), phenol red for detecting carbohydrate fermentation and ferrous sulfate for detection of hydrogen sulfide production (indicated by blackening in the butt of the tube) a differential medium that can distinguish between a number of Gram-negative enteric bacteria based on their physiological ability (or lack thereof) to: a. metabolize lactose and/or sucrose b. conduct fermentation to produce acid c. produce gas during fermentation d. generate H2S –

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INTERPRETATION OF RESULTS: A/A = yellow throughout K/A = red slant, yellow butt K/K = red or red/orange throughout carbon dioxide = bubbles or breaks in medium black precipitate = presence of hydrogen sulfide SLANT COLOR:

Code letter:

Interpretation

RED

R

does not ferment either lactose or sucrose

YELLOW

Y

ferments lactose and/or sucrose

SCORING THE BUTT COLOR AND CONDITION: Code BUTT Lette Interpretation COLOR/CONDITION r RED

YELLOW

R

no fermentation, the bacterium is an obligate aerobe

Y

some fermentation has occurred, acid has been produced, it is a facultative anaerobe.

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GAS FORMED

YG

Seen as cracks in the agar, bubbles, or the entire slant may be pushed out of the tube. (Caution:these gassy fermenters may have bacteria close to the opening.)

BLACK

"+"

H2S has been produced

Slant/Butt Reaction

Color(Slant/Bu Utilization Examples tt) Glucose, Pseudomon Alkaline/Alkalin lactose, Red/Red as e (K/K) sucrose not aeruginosa fermented Alkaline/Acid Glucose only Red/Yellow Shigella (K/A) fermented Glucose fermented, Escherichia Acid/Acid (A/A) Yellow/Yellow lactose+ orcoli, sucrose Klebsiella fermented Alkaline/Acid Blackening of Glucose onlyProteus, (K/A) with H2Smedium may be fermented Salmonella produced throughout tube

b. IMViC Test A. IMViC represents the first letter of each individual test within the series: 1. Indole test (tryptone broth) 2. Methyl Red (MR-VP broth) 3. Voges-Proskauer (MR-VP broth) 4. Citrate (Citrate agar slants) B. This series of tests has been used for many years to differentiate gram-negative enteric bacteria (Enterobacteriaceae). a. Pathogenic (Salmonella, Shigella) b. Occasionally pathogenic (Proteus, Klebsiella) c. Normal Flora (Escherichia, Enterobacter) Enterobacteriaeae (enterics) – are Gram-negative bacteria that grow in the intestinal tract of humans and other animals 21


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IMViC tests are frequently employed for identification of this group of microbes which includes such organisms as Klebsiella, Enterobacter, and Escherichia coli presence of E. coli is used by public health officials as an indicator of fecal contamination of food and water supplies Enterobacter and Klebsiella resemble E.coli in being lactose fermenters, their presence does not necessarily indicate fecal contamination because they are widespread in soil and grass

C. The significance of these tests is that when testing drinking water for the presence of the sewage indicator E. coli, one must be able to rule out Enterobacter aerogenes. E. aerogenes is not always associated with sewage, and its presence in water would not necessarily indicate sewage contamination. ============================================== ======================== Indole Methyl Red VP Citrate E. coli + + E. aerogenes + + ============================================== ======================== C. Results are recorded in the same order as the name sequence. For example: ++-- or --++. D. Individual tests of the IMViC: 1. Indole Test a. Tryptophane, an essential amino acid, can be oxidized by some organisms.

b. Test: SIM (Sulfide, Indole, Motility) Agar, which contains tryptophan, is used. Kovac’s reagent (P-Dimethlyaminobenzaldehyde) is added to growth. Organisms that produce indole yield a red layer at the top of tube (positive reaction). c. test organism is inoculated into tryptone broth, a rich source of the amino acid tryptophan

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d. indole positive bacteria such as Escherichia coli produce tryptophanase, an enzyme that cleaves tryptophan, producing indole and other products e. when Kovac's reagent (p-dimethylaminobenzaldehyde) is added to a broth with indole in it, a dark pink color develops f. indole test must be read by 48 hours of incubation because the indole can be further degraded if prolonged incubation occurs. The acidic pH produced by Escherichia coli limits its growth. g. Description: – tryptophan hydrolysis -Some bacteria split tryptophan into indole and pyruvic acid using the hydrolase called tryptophanase – can be detected with Kovac's reagent (Indole reagent) – very important in differentiating E. coli (indole positive) from some closely related enteric bacteria – also differentiates Proteus mirabilis (indole negative) from all other Proteus species (indole positive) h. Interpretation: After incubation: The broth must be turbid. A clear broth indicates that the organism did not grow and cannot be tested. Add a few drops of Indole reagent to the broth culture (tryptone broth). A positive result has a red layer at the top. A negative result has a yellow or brown layer. 2. Methyl Red Test a. qualitative test of acid produced from oxidation of glucose b. used primarily to differentiate E. coli from E. aerogenes – E. coli – produces large amounts of acid. – E. aerogenes -- produces neutral or non-acidic end products.

c. Test: Organism is grown in MR-VP broth. Methyl red indicator is added to culture to identify amount of acid present. When organism is E. coli, medium pH=4 and is red (positive). When acid is present, but at a much lower concentration, pH = 6, the medium turns yellow. d. contains glucose and peptone. All enterics oxidize glucose for energy; however the end products vary depending on bacterial enzymes. 23


e. used to determine what end products result when the test organism degrades glucose. E. coli is one of the bacteria that produces acids, causing the pH to drop below 4.4. When the pH indicator methyl red is added to this acidic broth it will be cherry red (a positive MR test). f. Klebsiella and Enterobacter produce more neutral products from glucose (e.g. ethyl alcohol, acetyl methyl carbinol). In this neutral pH the growth of the bacteria is not inhibited. The bacteria thus begin to attack the peptone in the broth, causing the pH to rise above 6.2. At this pH, methyl red indicator is a yellow color (a negative MR test). g. Description: – Mixed acid fermentation - Many gram-negative intestinal bacteria can be differentiated based on the products produced when they ferment the glucose in MR-VP medium. Escherichia, Salmonella, and Proteus ferment glucose to produce lactic, acetic, succinic, and formic acids and CO2, H2, and ethanol. The large amounts of acids produced lowers the pH of the medium - Methyl red (a pH indicator) will turn red when added to the medium if the organism was a mixed acid fermenter. Many of these organisms also produce gas. h. Interpretation: After incubation: The broth must be turbid. A clear broth indicates that your organism did not grow and cannot be tested. Remove 1 ml of broth and place into a sterile tube before performing the methyl red test if you are going to use the same broth for the VP test. Add 3-4 drops of methyl red to the original broth. A positive result has a distinct red layer at the top of the broth. A negative result has a yellow layer. 3. Voges-Proskauer Test a. Determines ability of an organism to produce non-acid or neutral end products from organic acids present following glucose metabolism. b. Test: Organism is grown in MR-VP broth. Barritt’s reagent is added to culture. Pink/red color indicates presence of acetylmethylcarbinol (positive reaction), a natural product formed from pyruvic acid in the course of glucose fermentation. c. contains glucose and peptone. All enterics oxidize glucose for energy; however the end products vary depending on bacterial enzymes. d. reagents used for the VP test are Barritt's A (alpha-napthol) and Barritt's B (potassium hydroxide). When these reagents 24


are added to a broth in which acetyl methyl carbinol is present, they turn a pink-burgundy color (a positive VP test). E. coli does not produce acetyl methyl carbinol, but Enterobacter and Klebsiella do. e. Principle: – demonstrate an organisms ability to convert pyruvate to acetoin. This property is a valuable tool in distinguishing Klebsiella, Enterobacter, Ewingella and Serratia from other members of Enterobacteraceae, as well as distinguishing between Proteus vulgaris (V-P negative) and P. mirabilis (V-P positive). 4. Citrate Test a. lactose is not present; organisms have two primary enzymes -citrate permease and citrase b. Test: Simmon’s Agar Slants are used. Bromthymol blue indicator is incorporated in the medium. If culture grows (citrate positive, pH 7.6) media turns blue. If no growth, medium is green (pH 6.9) and culture is citrate negative. c. utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source. Simmon's media contains bromthymol blue, a pH indicator with a range of 6.0 to 7.6. Bromthymol blue is yellow at acidic pH's (around 6), and gradually changes to blue at more alkaline pH's (around 7.6). Uninoculated Simmon's citrate agar has a pH of 6.9, so it is an intermediate green color. Growth of bacteria in the media leads to development of a Prussian blue color (positive citrate). Enterobacter and Klebsiella are citrate positive while E.coli is negative. – Thus E.coli gives ++-- results on the IMViC tests, while Enterobacter and Klebsiella give the reverse: --++ d. Description: – Simmon's citrate agar tests for the ability of an organism to use citrate as its sole source of carbon. This media contains a pH indicator called bromthymol blue. The agar media changes from green to blue at an alkaline pH. e. Interpretation: After incubation: A positive reaction is indicated by a slant with a Prussian blue color. A negative slant will have no growth of bacteria and will remain green. IMViC Tests

Results

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Indole Test Bacteria were grown in tryptone broth. The uninoculated tube is on the left. The positive indole test is in the center. The negative indole test is on the right.

Methyl Red (MR) Test Bacteria were grown broth. The uninoculated tube left. The positive MR test center. The negative MR test right.

in MRVP is on the is in the is on the

Voges-Praskauer (VP) Test Bacteria were grown in MRVP broth. The uninoculated tube is on the left. The positive VP test in the center. The negative VP test is on the right.

26


Citrate test Bacteria were grown on Citrate Agar slants. The uninoculated tube is on the left. The positive Citrate test in the center. The negative Citrate test is on the right.

Give the nutritive value of the different ingredients of nutrient agar. ??? No Answer Yet…???

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Cultivation of Microorganisms

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