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What is microbiology ?
Microbiology could be defined as the study of organisms too small to be seen with the naked eye. Figure 1.1 shows the relative size of microbes compared to other living things. However, the relatively recent discovery of bacteria of near 1 mm in size has made this definition somewhat inaccurate and in the grand tradition of science, a new definition is in order.

The chart above shows how microorganisms are related. The three most general groups into which the organisms are placed are prokaryotes, eukaryotes, and non-living organisms.

Figure 1.1 The relative size of microbes. Though microbes are small, they nevertheless span a large range of sizes from the smallest bacterial cells at ~0.15 m to giant bacteria larger than 700 m. The viruses depicted at the far left of the scale are even smaller.

We will consider microbiology to be the study of organisms that can exist as single cells, contain a nucleic acid genome for at least some part of their life cycle, and are capable of replicating that genome. This broad description encompasses an understandably large group of organisms including fungi, algae, protozoa and bacteria. This definition would also include viruses, which microbiology texts traditionally discuss along with living organisms.
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Microbiology also involves a collection of techniques to study and manipulate these small creatures. Because of their size, special instruments and methods had to be developed to allow the performance of interpretable experiments on microorganisms. These methods are not restricted to microbes alone, but have also found utility in working with populations of cells from higher organisms. Microorganisms are everywhere, but why are they worth learning about? The short answer is that they affect your life in many different ways. Before we begin our study of these creatures, we will first take a tour of some of their important habitats and point out why your existence depends upon them. We will then briefly explore the history of microbiology. If you ask the average person how microbes (or germs) impact their lives, they would immediately think of disease. This is not a silly view, as pathogenic microorganisms have greatly affected human populations throughout our existence. Until about 1930, microbes were the major cause of death in humans, with infectious disease infant mortality rates above 50%. From todays perspective this is a horrendous statistic, over half of all infants did not make it to adulthood! With the advent of antibiotics, vaccines and better water sanitation, humanity has reduced the impact of pathogenicmicrobes, but they will always remain an important social concern. The discipline of microbiology emerged from the study of these diseases and most advances in treating various ailments had their roots in this relatively young science. From the beginning of microbiology, significant resources have been spent to understand and fight disease-causing microorganisms. You may be surprised to learn that only a small fraction of microbes are involved in disease, many other microbes actually enhance our well being. In fact, like all other large organisms, humans are actually consortia of different organisms - there are more non-human cells in and on our bodies than there are human cells! Recent experiments, that have examined microorganisms inside our digestive tract by intensive sequencing experiments have revealed many interesting findings. More than 80% of the microbes in our guts have not been cultured. In addition, the microbial flora of a person is unique to that person, and there are differences based upon body type and genetic background. This has profound effects on physical wellbeing of the individual.

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Microbiological investigation


Macroscopic morphology (appearance of bacterial colonies on petri dish Microscopic morphology (bacterial shape & arrangement under the microscope) Physiological / biochemical characteristics (metabolism: aerobes vs anaerobes) Chemical analysis (cell wall composition) Serological analysis (antibodies) Genetic & molecular analysis (DNA & rRNA sequence)

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Identification plan for genus Staphylococcus

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Gram Positive Organisms

Aerobic, Gram-positive cocci
Staphylococcus aureus Staphylococcus epidermidis Staphylococcus sp. (Coagulase-negative) Streptococcus pneumoniae (Viridans group) Streptococcus agalactiae (group B) Streptococcus pyogenes (group A) Enterococcus sp.

Aerobic, Gram-positive rods

Bacillus anthracis Bacillus cereus Bifidobacterium bifidum Lactobacillus sp. Listeria monocytogenes Nocardia sp. Rhodococcus equi (coccobacillus) Erysipelothrix rhusiopathiae Corynebacterium diptheriae Propionibacterium acnes

Anaerobic, Gram-positive rods

Actinomyces sp. Clostridium botulinum Clostridium difficile Clostridium perfringens Clostridium tetani Mobiluncus sp. (gram-variable or gram-negative but has a gram-positive cell wall)

Anaerobic, Gram-positive cocci

Peptostreptococcus sp.

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Gram Negative Organisms

Aerobic, Gram-negative cocci
Neisseria gonorrhoeae Neisseria meningitidis Moraxella catarrhalis

Anaerobic, Gram-negative cocci

Veillonella sp.

Aerobic, Gram-negative rods

Fastidious, Gram-negative rods o Actinobacillus actinomycetemcomitans (fig1, 2) o Acinetobacter baumannii(fig 1 really A. calcoaceticus) o Bordetella pertussis (fig 1, 2) o Brucella sp. (fig 1) o Campylobacter sp.(fig 1, 2) o Capnocytophaga sp.(fig 1, 2) o Cardiobacterium hominis (fig 1) o Eikenella corrodens (fig 1) o Francisella tularensis (fig 1,) o Haemophilus ducreyi (fig 1, 2) o Haemophilus influenzae (fig 1, 2, 3) o Helicobacter pylori (fig 1, 2, 3) o Kingella kingae (fig 1) o Legionella pneumophila (fig 1, 2, 3) o Pasteurella multocida (fig 1, 2) o Klebsiella granulomatis (formerly calledCalymmatobacterium granulomatis (Gram negative rod)(fig 1) Enterobacteriaceae (glucose and lactose fermenting Gram-negative rods) o Citrobacter sp. (fig 1) o Enterobacter sp. (fig 1, 2) o Escherichia coli (fig 1, 2) o Klebsiella pneumoniae (fig 1, 2) Fermenting glucose but NOT lactose; Gram-negative rods o Proteus sp. (fig 1) o Salmonella enteriditis (fig 1) o Salmonella typhi (fig 1) o Shigella sp. (fig 1, 2) o Serratia marcescens (fig 1, 2) o Yersinia enterocolitica (fig 1) o Yersinia pestis (fig 1, 2) Oxidase-positive, glucose-fermenting Gram-negative rods o Aeromonas sp. (fig 1) o Plesiomonas shigelloides (fig 1) o Vibrio cholerae (fig 1, 2) o Vibrio parahaemolyticus (fig 1, 2) o Vibrio vulnificus (fig 1, 2) Glucose-nonfermenting, Gram-negative rods o Acinetobacter sp. (fig 1) o Flavobacterium sp. (fig 1) o Pseudomonas aeruginosa (fig 1, 2, 3) o Burkholderia cepacia (fig 1) o Burkholderia pseudomallei (fig 1, 2) o Xanthomonas maltophilia or Stenotrophomonas maltophila(fig 1)

Anaerobic, Gram-negative rods

Bacteroides fragilis (fig 1) Bacteroides sp. (fig 1) Prevotella sp. (fig 1) Fusobacterium sp. (fig 1,2, 3

Gram-negative spiral
Spirillum minus (minor)- (fig 1)

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Gram stain

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(purple dye)



(safranin as counterstain)

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Acid fast stain (Ziehl Neelsen stain)

Mycobacterium tuberculosis bacteria (Magnified 1000X). Acid fast organisms stain red. Non acid fast organisms and tissue cells stain blue.

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Spore stain

The Gram-positive Clostridium subterminale bacteria, which had been cultivated on a blood agar plate (BAP)

bacillus subtilis

Spore Stain of Bacillus megaterium

The cells in this figure were stained with malachite green to stain endospores and the vegetative cells were stained with safranin. The top arrow is pointing to a free endospore, the middle arrow is pointing to a red vegetative cell which does not have an endospore, and the lower arrow is pointing to a vegetative cell which still has the endospore with in it. The red outline of the cell is still visible but the endospore takes up most of the cell space.

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smear with malachite green

heat over the flame for 3 min. Keep the smear covered with the dye and dont allow to boil

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Escherichia coli (Gram-negative rods) Magnification: 1000

Escherichia coli (Gram-negative rods) Magnification: 1000

Klebsiella pneumoniae & Staphylococcus aureus Magnification: 1000 Gram-negative rods and gram-positive cocci

Moraxella catarrhalis Magnification: 1000 Gram-negative diplococci

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Most isolation media are a combination of two or more types listed above.
Name of Media Trypticase Soy Agar/Broth TSA, TSB Blood Agar BAP Type General All Purpose 1) To grow fastidious bacteria. Enrichment Differential 2) To differentiate between bacteria based on hemolysis. Use in Medical Lab Ingredients Growth Allowed Growth Inhibited Fastidious organisms such as Streptococcus

Basic nutrients

Most organisms

MacConkey Agar MAC

Selective Differential

Ex/ Streptococcus 1) To select for Gram negative enteric bateria. Bile Salts (Selective) Crystal Violet 2) To differentiate between Gram negative (Selective) Gram negative enteric coliforms, other Lactose (differential) bacteria based on lactose Gram negative Neutral Red (indicator) fermentation. Ex/ Escherichia coli 1) To select for salt tolerant Gram positive bacteria.

Beef heart (enrichment) Sheep RBCs (differential)

Most organisms including Streptococcus

Few or none

Gram positive

Mannitol Salt Agar MSA

Selective Differential

7.5% Salt (selective) 2) To differentiate between salt tolerant bacteria based on Mannitol (differential) Phenol red (indicator) mannitol fermentation. Ex/ Staphylococcus aureus

Staphylococcus, Bacillus, other Gram positive

Most other bacteria

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Chocolate agar (CA)

Use : For the isolation and cultivation of a variety of fastidious microorganism.

Microbiology agar plates (media for cultured bacteria)

CHOC is an enriched medium supplemented with cofactor, which provides NAD to facilitate the growth of Haemophilus influenzae, Neisseria gonorrhoeae and Neisseria meningitidis. Heated sheep blood is added to give the medium its chocolate appearance.

Thayer-Martin agar (TMA)

Use : To select for fastidious organisms, such as N. gonorrhoeae, in patient samples containing large numbers of normal flora, such as in the female genital tract

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MacConkey agar (MC)

Use : For the selective isolation, cultivation and differentiation of coliformsand enteric pathogens based on the ability to ferment lactose. Lactosefermening organisms appear as red to pink colonies. Lactose-nonfermenting organisms appear as colorless or transparent colonies

Blood agar (BA)

Use : For the isolation, cultivation and detection of hemolytic activity of streptococci, pneumococci and other particular fastidious microorganisms

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Salmonella-Shigella agar (SS)

Use : For the selective isolation and differentiation of pathogenic enteric bacilli, especially those belonging to the genus Salmonella. This media is notrecommended for the primary isolation of Shigella species. Lactose-fermenting bacteria such as Escherichia coli or Klebsiella pneumoniae appear as small pink or red colonies. Lactose-nonfermenting bacteria such as Salmonella species, Proteusspecies and Shigella species appear as colorless colonies. Production of H2S bySalmonella species turns the center the colonies black.

Eosin Methylene Blue agar (EMB)

Use : For the isolation, cultivation and differentiation of Gram-negative enteric bacteria based on lactose fermentation. Bacteria that ferment lactose, especially the coliform bacterium Escherichia coli, Appear as colonies with green metallic sheen or blueblack to brown color. Bacteria that do not ferment lactose appear as colorless or transparent light purple colonies

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Thiosulfate Citrate Bile Salt Sucrose agar (TCBS)

Use : For the selective isolation of Vibrio cholerae and Vibrio parahaemolyticus from a variety of clinical specimens and in epidemiological investigations.

Sabouraud Dextrose agar (SDA)

Use : For the cultivation of pathogenic and nonpathogenic fungi, especially dermatophytes. The medium may be made more selective for fungi by the addition of specific antibiotics such as chloramphenicol. For the cultivation of yeast and filamentous fungi.

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Tryptic Soy agar (TSA)

Use : Cultivation on non-fastidious bacteria

Mannitol Salt agar (MSA)

Use : Selects for Staphylococci, which grow at high salt concentrations, differentiates Staphylococcus aureus from other Staphylococci

Staphylococcus aureus

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Lowenstein-Jensen Media Agar for Mycobacterium

Mycobacterium tuberculosis colonies

Different mycobacteria species grown on TB-Medium Base according to Lwenstein- Jensen

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Mueller-Hinton agar (MHA)

Use : For antimicrobial susceptibility testing of a variety of nonfastidious, rapidly-growing microorganisms.

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Spirit Blue agar

Use : This agar is used to identify organisms that are

capable of producing the enzyme lipase. This enzyme is secreted and hydrolyzes triglycerides to glycerol and three long chain fatty acids. These compounds are small enough to pass through the bacterial cell wall. Glycerol can be converted into a glycolysis intermediate. The fatty acids can be catabolized and their fragments can eventually enter the Krebs cycle. Spirit blue agar contains an emulsion of olive oil and spirit blue dye. Bacteria that produce lipase will hydrolyze the olive oil and produce a halo around the bacterial growth. The Gram-positive rod, Bacillus subtilis is lipase positive (pictured on the right) The plate pictured on the left is lipase negative.

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Hemolytic bacteria


Alpha hemolysis: this is the partial destruction of red blood cells, and often has a greenish hue to it rather than an actual clearing around the colonies, Beta hemolysis: which is the complete destruction of red blood cells, usually, if you can see a finger or some other object through the clearing on the plate Gamma hemolysis: no hemolysis

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Biochemical identification

Tests used to identify Gram Positive Bacteria

Catalase Test Mannitol Salt Agar (MSA) Blood Agar Plates (BAP) Motility Agar Coagulase Test Taxos P (optochin sensitivity testing) Taxos A (bacitracin sensitivity testing) CAMP Test Bile Esculin Agar Nitrate Broth Spirit Blue agar Starch hydrolysis test

Tests used to identify Gram Negative Bacteria

Oxidase Test Sugar (eg glucose) broth with Durham tubes Methyl Red / Voges-Proskauer (MR/VP) Kligers Iron Agar (KIA) Nitrate Broth Motility Agar MacConkey agar Simmons Citrate Agar Urease test Sulfur Indole Motility Media (SIM)

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The Controls That Were Performed For The Various Tests

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Urease test
Certain bacteria produce the enzyme urease during its metabolism process and that will break down the urea in the medium to ammonia and carbon dioxide creates an alkaline environment that causes the phenol red to turn to deep pink. This is a positive reaction for the presence of urease. Failure of deep pink color to develop is evidence of a negative reaction

Indole Test
ndole tests looks for the presence or absence of tryptophanase enzyme production of the bacteria. If the enzyme is present,it will degrade the aminoacid tryptophan in the media and will produce Indole,ammonia and pyruvic acid. Indole will react with Kovacs reagent to produce a cherry red complex,which indicates a positive indole test. The absence of red color is indicative of tryptophan hydrolysis due to the lack of tryptophanse enzyme

Strong Urease produc tion

Uninoculated Negative Tube Urease producti on

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TSI (triple sugar iron)

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Result (slant/butt) 1 Red/Yellow 2 Yellow/Yellow 3 Red/Red 4 Yellow/Yellow with bubbles 5 Red/Yellow with bubbles 6 Red/Yellow with bubbles and black precipitate 7 Yellow/Yellow with bubbles and black precipitate 8 Red/Yellow with black precipitate 9 Yellow/Yellow with black precipitate
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Symbol K/A A/A K/K A/A,G K/A,G K/A,G,H2S A/A,G,H2S K/A,H2S A/A,H2S

Interpretation Glucose fermentation only, peptone catabolized. Glucose and lactose and/or sucrose fermentation. No fermentation, Peptone catabolized. Glucose and lactose and/or sucrose fermentation, Gas produced. Glucose fermentation only, Gas produced. Glucose fermentation only, Gas produced, H2S produced. Glucose and lactose and/or sucrose fermentation, Gas produced, H2S produced. Glucose fermentation only, H2S produced. Glucose and lactose and/or sucrose fermentation, H2S produced.
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DNAse Test

Note there is breakdown of the DNA in the agar. There is a clear zone (arrow) around the bacterial growth where there is no longer any DNA left in the agar to precipitate out of solution after the HCl was added. General Many bacteria have enzymes that break down nucleic acids. The bacteria can then use the resulting nucleotides to build up their own nucleic acids. DNase is such an enzyme, which thus hydrolyzes DNA. Existence of DNase is characteristic for certain species or strains of bacteria and can be used for typing. Method Presence of DNase can be determined by cultivation on an agar plate, which contains DNA. If the bacterium has DNase and if the bacteria are allowed to grow over night, the DNA will be hydrolyzed into the constituting nucleotides. Diluted hydrochloric acid (HCl) is then poured onto the plate and there will be a clear zone close to the colonies or the streak, because individual nucleotides are soluble in diluted HCl, but not DNA, which precipitates in the rest of the plate.

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The citrate test 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.
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Catalase production and activity can be detected by adding the substrate H2O2 to an appropriately incubated (18- to 24-hour) tryptic soy agar slant culture. Organisms which produce the enzyme break down the hydrogen peroxide, and the resulting O2 production produces bubbles in the reagent drop, indicating a positive test. Organisms lacking the cytochrome system also lack the catalase enzyme and are unable to break down hydrogen peroxide, into O2 and water and are catalase negative

Catalase positive

Catalase negative

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Staphylococcus aureus

Staphylococcus epidermidis

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Gelatin hydrolysis tes

It is broken down by gelatinase into smaller polypeptides, peptones and amino acids that can cross the cell membrane and be utilised by the organism. when gelatin is broken down via hydrolysis, it cannotsolidify anymore, the areas of solid gelatin media where the organsim grows, will turn into liquid. Even if you refrigerate this medium, the media will remains liquid. Bacillus subtilisis able to produce proteolytic exoenzyme gelatinase to give the (+) result.

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This test detects the ability of microorganism to ferment glucose and to produce acidic end products. Enteric organism produces pyruvic acid from glucose metabolism. Some enteric will thn use the Mixed acid pathway to metabolize pyruvic acid to other acidic products such as lactic acid,acetic acid and formic acids. This will reduce the pH of the media. Methyl red is a pH indicator which is red at the acidic pH (below 4.4) and yellow at alkaline pH( above 7). The formation of red color after the addition of Methyl red reagent indicates the accumulation of acidic end products in the medium and is an indicative of positive test
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Susceptibility test

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Depicted above are two methods for determining antimicrobial susceptibility of group B Streptococcus (GBS). It is especially important that the microbiologist determine the susceptibility of GBS to erythromycin and clindamycin as these two drugs are used as substitutes if the patient cannot be treated with penicillin. The long strip on the left is called the E-testTM. This picture shows the drug erythromycin (EM). In this test, the strip contains a gradation of antibiotic with the strongest being at the top of the strip and the weakest at the bottom. The minimum inhibitory concentration (MIC) of antibiotic that will inhibit the bacteria is determined by where the growth of the bacteria starts, or the area of the growth where the ellipse growth meets the strip.

For this example the MIC for erythromycin is 0.19 g/ml which is considered to be susceptible based on CLSI breakpoints. Consult the latest CLSI manual* for interpretation of MICs for Streptococcusspp. Beta-hemolytic Group (2010 breakpoints): Clindamycin: 0.25 g/ml = susceptible, 0.5 g/ml = intermediate, and 1.0 g/ml = resistant; and for Erythromycin: 0.25 g/ml = susceptible, 0.5 g/ml = intermediate, and 1.0 g/ml = resistant. The two disks on the right of the agar plate are erythromycin (E) and clindamycin (DA). There are criteria for the zones of inhibition of growth that determine whether or not the bacteria are susceptible or resistant. In the test shown above, the bacterium is susceptible to erythromycin (the zone is 21 mm) and susceptible to clindamycin (zone is 19 mm). The disk test is perfectly satisfactory for determining the antimicrobial susceptibility of GBS. Interpret according to CLSI guidelines for Streptococcus spp. Beta-hemolytic Group (2010 breakpoints for diskdiffusion: for clindamycin: 19 mm = susceptible, 1618 mm = intermediate, and 15 mm = resistant; for erythromycin: 21 mm = susceptible, 1620 mm = intermediate, and 15 = resistant.
*Clinical and Laboratory Standards Institute. Performance standard for antimicrobial susceptibility testing, M100-S20 (2010), M-2 and M-7, Wayne, PA.

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Taxos P (optochin sensitivity testing)

Taxos A (bacitracin sensitivity testing)

This is a differential test used to distinguish between organisms sensitive to the antibiotic optochin and those not. This test is used to distinguishStreptococcus pneumoniae (optochin sensitive (pictured on the right below)) from other a-hemolytic streptococci (optochin resistant (Streptococcus mitis is pictured on the left below).

This is a differential test used to distinguish between organisms sensitive to the antibiotic bacitracin and those not. Bacitracin is a peptide antibiotic produced by Bacillus subtilis. It inhibits cell wall synthesis and disrupts the cell membrane. This test is commonly used to distinguish between the-hemolytic streptococci: Streptococcus agalactiae (bacitracin resistant) and Streptococcus pyogenes (bacitracin sensitive). The plate below was streaked with Streptococcus pyogenes; notice the large zone of inhibition surrounding the disk.

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Carbohydrate fermentation

The identification of some bacteria is aided by determining what nutrients the bacteria can utilize and what end products will be produced in the process. These characteristics are controlled by the enzymes which the bacteria produce. Because the type of enzyme(s) bacteria produce is genetically controlled, the pattern of sugars fermented may be unique to a particular species or strain. Fermentation products are usually acid (lactic acid, acetic acid etc.), neutral (ethyl alcohol etc.), or gases (carbon dioxide, hyrogen, etc.). A bright yellow color indicates the production of enough acid products from fermentation of the sugar to drop the pH to 6.9 or less. Production of gas is determined with a Durham tube, a small inverted vial filled with the carbohydrate fermentation broth. If gas is produced during fermentation of the sugar, it is trapped at the top of the Durham tube and appears as a bubble.

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Table of durham fermentation

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If the laboratory is not able to identify group B streptococci (GBS) by the Lancefield grouping procedure, there are other microbiologic tests that can be used to identify GBS. This picture shows one of these tests. It is called the CAMP test. CAMP is an acronym for the authors of this test (Christie, Atkinson, Munch, Peterson). The CAMP test takes advantage of the capacity of GBS to produce this CAMP factor; most other hemolytic streptococci do not produce CAMP factor. This picture shows the group B Streptococcus (on the right) and a group A Streptococcus (GAS) (on the left). Down the middle we have inoculated the plate with a Staphylococcus aureus strain (vertical streak). We then inoculated the GBS (on right) and GAS (on left) perpendicular to the Staphylococcus streak. We inoculated the agar plate so as not to touch the two different organisms (Staphylococcusand Streptococcus) but to come close to each other. TheStaphylococcus is used because it produces a lysin that only partially lyses the red blood cells (called beta-lysin). The CAMP factor reacts with the partially lysed area of the blood agar plate to enhance the hemolytic activity. Note the arrowhead shape of the zone of enhanced hemolytic activity by the GBS near the Staphylococcus streak (on right) but not by the GAS (on left). This means that the bacterium on the right is GBS because it is producing a CAMP factor.

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Oxidative-Fermentative Metabolism

ecoli, eaerogenes (controls included) OF test

How do we determine if type of metabolism is fermentative or oxidative? we will test with a glucose OF oxidative- fermentative agar. If agar turns yellow, acid is produced. If agar turns green, no acid is produced. Motility of the bacteria can also be determined by the presence of turbidity (cloudiness) OXIDATIVE metabolism may or may not cause an acid to be produced for aerobic conditions. No acid will be produced for anaerobic conditions. FERMENTATIVE metabolism will cause an acid to be produced for aerboic and anaerobic conditions.

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Oxidative-Fermentative Metabolism
OF test(yellow-acid, green-no acid)

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Protein Catabolism (Proteolysis)

Casein(a protein) is broken down by protease into peptones and amino acids. During the degradation process, polypeptide bonds are broken. Once the bonds are broken, amino acids are produced. A clear zonesurrounding streak line of agar indicates a (+) result.
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Voges Proskauer Test

This test determines the ability of microorganism to ferment glucose. The end products of glucose metabolism,pyruvic acid, is further metabolized by using Butylene glucol pathway to produce neutral end such as acetoin and 2,3 butanediol. When Barrits reagent A ( 40% KOH) and Barrits reagent B(5% solution of alpha naphthol) is added it will detect the presence of acetoin,the precursor in the 2,3- butanediol synthesis. Acetoin in the presence of Oxygen and Barrits reagent is oxidized to diacetyl. in the presence of alpha naphthol catalyst. Diacetyl then reacts with guanidine components of peptone to produce a cherry red color

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This is a differential medium. It is used to determine if an organism is capable of reducing nitrate (NO3-) to nitrite (NO2-) or other nitrogenous compounds via the action of the enzyme nitratase (also called nitrate reductase). This test is important in the identification of both Gram-positive and Gram-negative species. After incubation, these tubes are first inspected for the presence of gas in the Durham tube. In the case of nonfermenters, this is indicative of reduction of nitrate to nitrogen gas. However, in many cases gas is produced by fermentation and further testing is necessary to determine if reduction of nitrate has occurred. This further testing includes the addition of sulfanilic acid (often called nitrate I) and dimethyl-alpha-napthalamine (nitrate II). If nitrite is present in the media, then it will react with nitrate I and nitrate II to form a red compound. This is considered a positive result. If no red color forms upon addition of nitrate I and II, this indicates that either the NO3- has not been converted to NO2- (a negative result), or that NO3- was converted to NO2- and then immediately reduced to some other, undetectable form of nitrogen (also a positive result). In order to determine which of the preceding is the case, elemental zinc is added to the broth. Zinc will convert any remaining NO3- to NO2- thus allowing nitrate I and nitrate II to react with the NO2- and form the red pigment (a verified negative result). If no color change occurs upon addition of zinc then this means that the NO3- was converted to NO2and then was converted to some other undetectable form of nitrogen (a positive result). If the nitrate broth turns red (tubes pictured in the center) after nitrate I and nitrate II are added, this color indicates a positive result. If instead, the tube turns red (tube pictured on the left) after the addition of Zn, this indicates a negative result. If there is no color change in the tube after the addition of nitrate I and nitrate II, the result is uncertain. If the tube is colorless (picture on the right) after the addition of Zn this indicates a positive test. Fakultas Kedokteran UNISBA

Nitrate test

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Bile esculin test

Bile Esculin Agar is recommended for the presumptive isolation and identification of group D streptococci. Bile Esculin Agar contains esculin, ferric citrate and 4% oxgall. This concentration of oxgall will inhibit practically all strains ofstreptococci other than group D. If an organism growing on the medium hydrolyzes esculin, a glycone is formed. This compound will react with the ferric ions in the medium forming a black coloration in the medium.

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Bile solubility test

Use : To differentiates Streptococcus pneumoniae from other -hemolytic streptococci by demonstrating its susceptibility to lysis in the presence of bile. When a bile salt such as desoxycholate is added directly to Streptococcus pneumoniae growing on an agar plate or in a broth culture the bacteria will lyse and the area become clear. Other alpha-hemolytic streptococci are resistant to (not lysed by) bile and will stay visible or turbid (cloudy)

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Starch hydrolysis test

This test is used to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the enzymes -amylase and oligo-1,6-glucosidase. Often used to differentiate species from the genera Clostridium and Bacillus. Because of the large size of amylose and amylopectin molecules, these organisms can not pass through the bacterial cell wall. In order to use these starches as a carbon source, bacteria must secrete-amylase and oligo1,6-glucosidase into the extracellular space. These enzymes break the starch molecules into smaller glucose subunits which can then enter directly into the glycolytic pathway. In order to interpret the results of the starch hydrolysis test, iodine must be added to the agar. The iodine reacts with the starch to form a dark brown color. Thus, hydrolysis of the starch will create a clear zone around the bacterial growth.Bacillus subtilis is positive for starch hydrolysis (pictured on the left). The organism shown on the right is negative for starch hydrolysis.

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Litmus milk test

Use: To differentiate bacteria based on various reactions that occur in skim milk suplemented with a litmus pH indicator. Principle: Milk is a complex nutritional source that contains proteins (mainly casein) in an aqueous solution of lactose and minerals. Bacterial enzymes alter the media and may bring about various changes. Litmus is added to the medium to detect pH changes that may occur as a result of these enzymatic reactions. Above a pH of 8.3 litmus is blue, while below a pH of 4.5 litmus is red.

Litmus milk 1 control, 2 pink = acid, 3 white = reduction, 4 & 5 stormy fermentation, 6 blue = alkaline
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Microbiology : Biochemical Tests (Review)

Test Amylase Caseinase DNase Lipase Medium starch agar skim milk agar DNase agar spirit blue agar Substrate starch casein ds DNA lipid (triglyceride) Exoenzymes Product(s) Reagent glucose, maltose, dextrins iodine added after incubation amino acids, peptides nucleotides glycerol, fatty acids methyl green in agar (bound to DNA) spirit blue in agar Result + = colorless zone around organism after iodine + = clear zone around organism + = colorless zone around organism + = blue/clear zone around organisms, blue ppt. in organisms

Test EMB agar selective for gm - & differential for lactose fermentation MacConkeys agar selective for gm - & differential for lactose fermentation Mannitol Salt Agar (MSA) selective for staph & differential for mannitol fermentation

Medium Eosin Methylene Blue (EMB) agar MacConkeys agar



Selective and Differential Media Selective pH Result Ingredient indicator Growth = gm -; Dye accumulation (color) in organism Eosin + = lactose fermentation: Methylene Blue Metallic green Fish eyes (pink with purple center) Crystal violet + bile salts Neutral red in agar Growth = gm -; Pink ppt. in organism = lactose fermentation Growth = salt tolerant (staphylococci) Acidic (yellow) media = mannitol fermentation

+ organisms

E. coli E. aerogenes


E. coli; E. aerogenes S. epidermidis & S. aureus; S. aureus



7.5% NaCl

Phenol red in agar

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Test Indole Methyl Red Voges-Proskauer Citrate Utilization

Media Tryptic Soy Broth (TSB) MRVP broth MRVP broth Simmons citrate agar

Substrate Tryptophan Glucose Glucose Citrate

IMViC Product(s) Reagents Indole Kovacs layered on top Organic acids Methyl red Non-acids VP-A & VP-B (Barritts) Na2CO3

pH indicator (Methyl red ) Bromthymol blue in agar

+ appearance Red at surface Red throughout Red throughout Royal blue

+ organisms E. coli E. coli E. aerogenes E. aerogenes

Test H2S Gas Carbohydrate Fermentation

Media TSI agar (stab & drag) TSI agar (stab & drag)

Substrate Cysteine Glucose, lactose, sucrose

Product(s) H2S Organic acids

TSI (Triple sugar iron) Reagent pH indicator Iron in media Phenol red in agar

+ appearance Black ppt. when H2S reacts with iron to form iron sulfide Media: yellow = acidic (A); red = alkaline (K); Record reactions as slant/butt: K/A=glucose only, A/A=sucrose &/or lactose +/-glucose K/K=none fermented Black butt = acidic

+ organisms S. typhimurium P. vulgaris

Test Carbohydrate Fermentation Gas Production Test Urease Litmus

Media Carbohydrate broth with Durham tubes

Carbohydrate Fermentation Series Substrate Product(s) Glucose, lactose, or sucrose Organic acids CO2 Product(s) Ammonia, CO2 Various pH indicator Phenol red in agar Litmus in media

pH indicator Phenol red in broth

+ appearance Acidic (yellow) media Bubble in Durham tube + organisms Proteus

Media (Christensens) urea agar Litmus milk

Substrate Urea Casein, lactose

+ appearance pink (alkaline) media pink = acid; sugar fermentation hard curd = acid soft curd = rennin clots casein blue = alkaline; casein breakdown brown = further casein breakdown white = litmus reduction

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Test Catalase Coagulase (Cytochrome) Oxidase Bile Solubility

Substrate Hydrogen peroxide (H2O2) Fibrinogen Cytochrome c

Product(s) Water, oxygen Clumping factor-fibrinogen complex Oxidized cytochrome c


+ appearance Bubbles Blue ppt. (clumping) Blue color Clearing (lysis of cells)

+ organisms Staphylococci S. aureus P. aeruginosa S. pneumoniae

Staphyloside latex beads Phenylenediamine Tris buffer + deoxycholate

Test *Alpha

Media Blood agar (5% SB)

Substrate RBCs & hemoglobin (Hb)

Hemolysis Results Hemolysis; partial digestion of Hb

+ appearance Green/brown zone around/beneath organisms Clear zone around organisms


Blood agar (5% SB)

RBCs & hemoglobin (Hb)

Hemolysis; complete digestion of Hb

+ organisms Strep. pneumoniae E.(S) faecalis Staph. epidermidis Strep. pyogenes, E.(S) faecium Staph. aureus

Blood agar (5% SB)

RBCs & hemoglobin (Hb)

No hemolysis

No clearing around organisms

*Alpha-hemolytic Strep differentiated by optochin sensitivity (S. pneumoniae) **Beta- hemolytic Strep differentiated by bacitracin sensitivity (S. pyogenes)

Test Nitrate Reduction

Media Nitrate broth

Substrate Nitrate

Product(s) Nitrite; N2/ammonia

Reagent Nitrate A & B, zinc added after incubation

Appearance 1. Add nitrate A & B: Red color = nitrate reductase 2. If colorless after nitrate A & B add zinc: Still colorless after zinc = both nitrate and nitrite reductase If red after zinc = neither nitrate or nitrite reductase Nitrite reductase

Nitrate reductase Nitrate (NO3) Nitrate A & B + nitrate = colorless Zinc reduces nitrate to nitrite Nitrite (NO2) Nitrate A & B + nitrite = red

N2 (gas) /NH3 (ammonia) Nitrate A & B + N2 (gas) or NH3 (ammonia) = colorless

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Bacterial Infection (Secondary Infection) (sign / symptoms) (Etiologic agent) (Site of Infection)

CLINICAL SPECIMENS (kind of specimens)

GRAM POSITIVE COCCI Blood agar (Measures of colony, Reaction on blood agar)

GRAM NEGATIVE ROD Mac Conkey agar (Morphology of colony, Reaction on M Conkey agar)

Lactose fermenter (Escherichia coli, Klebsiella pneumoniaea-Enterobacter) Catalase

Non-Lactose fermenter (Salmonella thypi, Shigella flexneri)

Catalase (-) (Streptococcus sp.) S. Pneumoniae (Optochin +, Inulin +) S. Viridans S. Hemolyticus

Catalase (+) (Staphylococcus sp.) S. aureus (Coagulase +) S. epidermidis (Novobiocin +) S. saprophyticus

KIA, MIU, Citrate

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Gram positive cocci

Sheep blood agar: Beta-hemolytic colonies indicate Staphylococcus aureus Mannitol Salt Agar Yellow colonies Differential test for mannitol fermentors and non-fermentators; S. aureus ferments mannitol. Catalase test Positive DNA-se test Positive Coagulase test Positive Coagulase test differentiates S. aureus, from other staphylococcus species such as S. epidermidis present in the normal flora.
A Gram stain of Staphylococcus aureus (Gram positive cocci, purple). grapelike cluster of stphylococcus

Antibiotic Susceptibility Tests Vancomycin, Amoxicillin with clavulanic acid, Cloxacillin, Ampicillin, Cephalosporon

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Staphylococcus is a very well known genus of bacteria. Colonies are "gold," or yellow on sheep blood agar solid media, hence the name. A common pathogen, boils, acne, wound infections, food poisoning are among a host of conditions caused by this organism. The organism is both pathogenic and invasive. It produces leukotoxin which can kill white blood cells and a wide variety of other toxins. S. aureus is quite pyogenic and in decades past was named Staphylococcus pyogenes, however that specific name is currently applied to one GPC, Streptococcus pyogenes.

S. aureus on mannitol agar plate

S. aureus on blood agar plate

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DNA-se test

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Gram negative - rod

McConkey agar Lactose fermenter, mucous Indole test Positive Methyl red test Positive Voges-Proskauer reaction test Positive Citrate utilization test Positive Urease test Positive Antibiotic Susceptibility Tests Penicillin, Cephalosporin, Aminoglycoside, Cephalosporin (e.g. Cefotaxime)
This inoculated MacConkey agar culture plate cultivated colonial growth of Gram-negative, small rod-shaped and facultatively anaerobic

String test of a Klebsiella pneumoniae isolate demonstrating hypermucoviscosity

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Gram Stain of Sputum Specimen Showing Capsules Surrounding a Gram Negative Bacillus

Cut surface of normal lung

Pneumonia caused by Klebsiella pneumoniae. K. pneumoniae can cause the disease Klebsiella pneumonia. They cause destructive changes to human lungs inflammation and hemorrhage with cell death (necrosis) that sometimes produces a thick, bloody, mucoid sputum (currant jelly sputum). Typically these bacteria gain access after a person aspirates colonizing oropharyngeal microbes into the lower respiratory tract.

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Urease test

Klebsiella pneumoniae biochemical test : Indole positive Urease positive Citrate positive Voges-Proskaueur positive TSI : yellow/yellow, gas (A/A,G) Positive citrate Negative citrate

Indole test
uninoculated A/A,G

Tripple Sugar Iron Medium (TSI medium) Glucose and lactose and/or Yellow/Yellow (acidic) A/A,G sucrose fermentation, Gas with bubbles produced. Fakultas Kedokteran UNISBA Page 76

References :
Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education Alfred.E.Brown. (2007). Bensonss microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill Levinson, W. (2006). Review of Medical Microbiology and Immunology. San Francisco,California: Lange Medical Books/ McGraw-Hill Medical Publishing Division>search

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