Download - Microbiology Guide FK-UNISBA
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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.
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.
What is microbiology ?
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 µm to giant bacteria
larger than 700 µm. The viruses depicted at the far left of the scale are even smaller.
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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 today’s 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 well-being of the individual. http://www.microbiologytext.com/index.php?module=Book&func=displayarticle&art_id=643
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Microbiological investigation
METHODS OF BACTERIAL IDENTIFICATION
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)
(mordant)
alcohol
(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
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.
The Gram-positive Clostridium subterminale bacteria, which had been cultivated on a blood agar plate (BAP)
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smear with malachite green
heat over the flame for 3 min. Keep the smear covered with the dye and don’t allow to boil
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Escherichia coli (Gram-negative rods)
Magnification: 1000×
Klebsiella pneumoniae & Staphylococcus aureus
Magnification: 1000× Gram-negative rods and gram-positive cocci
Escherichia coli (Gram-negative rods)
Magnification: 1000×
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
Type Use in Medical Lab Ingredients Growth Allowed Growth Inhibited
Trypticase Soy Agar/Broth TSA, TSB
General All Purpose
Basic nutrients Most organisms Fastidious organisms such as Streptococcus
Blood Agar BAP
Enrichment Differential
1) To grow fastidious bacteria.
2) To differentiate between bacteria based on hemolysis. Ex/ Streptococcus
Beef heart (enrichment) Sheep RBCs (differential)
Most organisms including Streptococcus
Few or none
MacConkey Agar MAC
Selective Differential
1) To select for Gram negative enteric bateria.
2) To differentiate between Gram negative enteric bacteria based on lactose fermentation. Ex/ Escherichia coli
Bile Salts (Selective) Crystal Violet (Selective) Lactose (differential) Neutral Red (indicator)
Gram negative coliforms, other Gram negative
Gram positive
Mannitol Salt Agar MSA
Selective Differential
1) To select for salt tolerant Gram positive bacteria.
2) To differentiate between salt tolerant bacteria based on mannitol fermentation. Ex/ Staphylococcus aureus
7.5% Salt (selective) Mannitol (differential) Phenol red (indicator)
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.
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
Microbiology agar plates
(media for cultured bacteria)
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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. Lactose–fermening 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 blue–black 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 Löwenstein- 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 Kreb’s 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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Hemolysis
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
Hemolytic bacteria
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Hemolysis
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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)
Kliger’s Iron Agar (KIA) Nitrate Broth Motility Agar
MacConkey agar Simmon’s Citrate Agar
Urease test Sulfur Indole Motility Media (SIM)
Biochemical identification
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The Controls That Were Performed
For The Various Tests
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Urease test
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 Kovac’s 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
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
Strong Urease production
Uninoculated Tube
Negative Urease production
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TSI (triple sugar iron)
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Result (slant/butt) Symbol Interpretation
1 Red/Yellow K/A Glucose fermentation only, peptone catabolized.
2 Yellow/Yellow A/A Glucose and lactose and/or sucrose fermentation.
3 Red/Red K/K No fermentation, Peptone catabolized.
4 Yellow/Yellow with bubbles A/A,G Glucose and lactose and/or sucrose fermentation, Gas produced.
5 Red/Yellow with bubbles K/A,G Glucose fermentation only, Gas produced.
6 Red/Yellow with bubbles and black precipitate K/A,G,H2S Glucose fermentation only, Gas produced, H2S produced.
7 Yellow/Yellow with bubbles and black precipitate A/A,G,H2S Glucose and lactose and/or sucrose fermentation, Gas produced, H2S produced.
8 Red/Yellow with black precipitate K/A,H2S Glucose fermentation only, H2S produced.
9 Yellow/Yellow with black precipitate A/A,H2S Glucose and lactose and/or sucrose fermentation, H2S produced.
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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.
DNAse Test
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CITRATE TEST
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 TEST (OXIDATIVE TEST)
Catalase positive Catalase negative
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
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COAGULASE TEST
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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METHYL RED TEST
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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For this example the MIC for erythromycin is 0.19 µg/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 μg/ml = susceptible, 0.5
μg/ml = intermediate, and ≥1.0 μg/ml = resistant; and for Erythromycin: ≤0.25 μg/ml = susceptible, 0.5 μg/ml = intermediate, and ≥1.0 μg/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 disk-
diffusion: for clindamycin: ≥19 mm = susceptible, 16–18 mm = intermediate, and ≤15 mm = resistant; for erythromycin: ≥21 mm = susceptible,
16–20 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.
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.
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Taxos A (bacitracin sensitivity testing)
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.
Taxos P (optochin 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).
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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.
Carbohydrate fermentation
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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.
CAMP TEST
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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 Barrit’s reagent A ( 40% KOH) and Barrit’s 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 Barrit’s 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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Nitrate test
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 NO2-
and 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.
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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 α-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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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 oligo-1,6-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.
Starch hydrolysis test
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Litmus milk 1 control, 2 pink = acid, 3 white = reduction, 4 & 5 stormy fermentation, 6 blue = alkaline
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.
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Microbiology : Biochemical Tests (Review)
Exoenzymes
Test Medium Substrate Product(s) Reagent Result
Amylase starch agar starch glucose, maltose, dextrins iodine added after incubation + = colorless zone around organism after iodine
Caseinase skim milk agar casein amino acids, peptides
+ = clear zone around organism
DNase DNase agar ds DNA nucleotides methyl green in agar (bound to DNA)
+ = colorless zone around organism
Lipase spirit blue agar lipid (triglyceride)
glycerol, fatty acids
spirit blue in agar + = blue/clear zone around organisms, blue ppt. in organisms
Selective and Differential Media
Test Medium Substrate Selective Ingredient
pH indicator
Result + organisms
EMB agar – selective for gm - & differential for lactose fermentation
Eosin Methylene Blue (EMB) agar
Lactose Eosin + Methylene Blue
Growth = gm -; Dye accumulation (color) in organism = lactose fermentation: Metallic green Fish eyes (pink with purple center)
E. coli E. aerogenes
MacConkey’s agar – selective for gm - & differential for lactose fermentation
MacConkey’s agar
Lactose Crystal violet + bile salts
Neutral red in agar
Growth = gm -; Pink ppt. in organism = lactose fermentation
E. coli; E. aerogenes
Mannitol Salt Agar (MSA) – selective for staph & differential for mannitol fermentation
MSA Mannitol 7.5% NaCl Phenol red in agar
Growth = salt tolerant (staphylococci) Acidic (yellow) media = mannitol fermentation
S. epidermidis & S. aureus; S. aureus
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IMViC
Test Media Substrate Product(s) Reagents pH indicator + appearance + organisms
Indole Tryptic Soy Broth (TSB) Tryptophan Indole Kovac’s layered on top Red at surface E. coli
Methyl Red MRVP broth Glucose Organic acids Methyl red (Methyl red ) Red throughout E. coli
Voges-Proskauer MRVP broth Glucose Non-acids VP-A & VP-B (Barritt’s) Red throughout E. aerogenes
Citrate Utilization Simmons’ citrate agar Citrate Na2CO3 Bromthymol blue in agar
Royal blue E. aerogenes
TSI (Triple sugar iron)
Test Media Substrate Product(s) Reagent pH indicator + appearance + organisms
H2S Gas TSI agar (stab & drag)
Cysteine H2S Iron in media Black ppt. when H2S reacts with iron to form iron sulfide
S. typhimurium P. vulgaris
Carbohydrate Fermentation
TSI agar (stab & drag)
Glucose, lactose, sucrose
Organic acids Phenol red in agar
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
Carbohydrate Fermentation Series
Test Media Substrate Product(s) pH indicator + appearance
Carbohydrate Fermentation Carbohydrate broth with Durham tubes
Glucose, lactose, or sucrose Organic acids Phenol red in broth Acidic (yellow) media
Gas Production CO2 Bubble in Durham tube
Test Media Substrate Product(s) pH indicator + appearance + organisms
Urease (Christensen’s) urea agar Urea Ammonia, CO2 Phenol red in agar pink (alkaline) media Proteus
Litmus Litmus milk Casein, lactose Various Litmus in 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 Substrate Product(s) Reagent + appearance + organisms
Catalase Hydrogen peroxide (H2O2)
Water, oxygen Bubbles Staphylococci
Coagulase Fibrinogen Clumping factor-fibrinogen complex Staphyloside latex beads Blue ppt. (clumping) S. aureus
(Cytochrome)Oxidase
Cytochrome c Oxidized cytochrome c Phenylenediamine Blue color P. aeruginosa
Bile Solubility Tris buffer + deoxycholate Clearing (lysis of cells) S. pneumoniae
Hemolysis
Test Media Substrate Results + appearance + organisms
*Alpha Blood agar (5% SB) RBCs & hemoglobin (Hb) Hemolysis; partial digestion of Hb Green/brown zone around/beneath organisms
Strep. pneumoniae E.(S) faecalis Staph. epidermidis
**Beta Blood agar (5% SB) RBCs & hemoglobin (Hb) Hemolysis; complete digestion of Hb Clear zone around organisms Strep. pyogenes, E.(S) faecium Staph. aureus
Gamma 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 Media Substrate Product(s) Reagent Appearance
Nitrate Reduction Nitrate broth Nitrate Nitrite; N2/ammonia
Nitrate A & B, zinc added after incubation
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
Nitrate reductase Nitrite reductase
Nitrate (NO3) Nitrite (NO2) N2 (gas) /NH3 (ammonia)
Nitrate A & B + nitrate = colorless
Zinc reduces nitrate to nitrite
Nitrate A & B + nitrite = red
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 GRAM NEGATIVE ROD Blood agar Mac Conkey agar
(Measures of colony, Reaction on blood agar) (Morphology of colony, Reaction on M Conkey agar)
Lactose fermenter Non-Lactose fermenter (Escherichia coli, (Salmonella thypi, Klebsiella pneumoniaea-Enterobacter) Shigella flexneri) Catalase
Catalase (-) Catalase (+) (Streptococcus sp.) (Staphylococcus sp.) S. Pneumoniae (Optochin +, Inulin +) S. aureus (Coagulase +) S. Viridans S. epidermidis (Novobiocin +) S. Hemolyticus S. saprophyticus
KIA, MIU, Citrate
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Gram positive cocci
A Gram stain of Staphylococcus aureus (Gram positive cocci, purple).
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. Antibiotic Susceptibility Tests Vancomycin, Amoxicillin with clavulanic acid, Cloxacillin, Ampicillin, Cephalosporon
grapelike cluster of stphylococcus
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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 blood agar plate
S. aureus on mannitol agar plate
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DNA-se test
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Enterobacteriaceae
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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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.
Gram Stain of Sputum Specimen Showing Capsules Surrounding a Gram Negative Bacillus
Cut surface of normal lung
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Tripple Sugar Iron Medium (TSI medium)
Yellow/Yellow (acidic) with bubbles
A/A,G Glucose and lactose and/or sucrose fermentation, Gas produced.
Klebsiella pneumoniae biochemical test : Indole positive Urease positive Citrate positive Voges-Proskaueur positive TSI : yellow/yellow, gas (A/A,G)
uninoculated A/A,G
Positive citrate
Negativecitrate
Urease test
Indole test
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References : Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education Alfred.E.Brown. (2007). Bensons’s 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 http://en.wikipedia.org/wiki/Main_Page http://www.textbookofbacteriology.net>search http://www.cdc.gov/groupbstrep/guidelines/slidesets.html http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm http://microblog.me.uk/ http://student.ccbcmd.edu/~gkaiser/ http://www.spjc.edu/hec/VT/VTDE/ATE2639LGS/gramstain.htm http://www.pc.maricopa.edu/Biology/rcotter/BIO%20205/LessonBuilders/Chapter%204%20LB/Ch4Lessonbuilder10.html http://faculty.mc3.edu/jearl/ML/index.html http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/dental/tutorials/flora/Gram.html http://www.slic2.wsu.edu:82/hurlbert/micro101/pages/101lab6.html http://lifesci.rutgers.edu/genmicrolab/index.htm http://amrita.vlab.co.in/?sub=3&brch=76&sim=1109&cnt=1 http://web.med.unsw.edu.au/cdstest/ etc.
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