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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.
http://www.microbiologytext.com/index.php?module=Book&func=displayarticle&art_id=643
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Microbiological investigation
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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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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
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heat over the flame for 3 min. Keep the smear covered with the dye and dont allow to boil
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Klebsiella pneumoniae & Staphylococcus aureus Magnification: 1000 Gram-negative rods and gram-positive cocci
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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
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.
Few or none
Gram positive
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
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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.
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Staphylococcus aureus
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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
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
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Hemolysis
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Biochemical identification
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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
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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
Fakultas Kedokteran UNISBA
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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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 positive
Catalase negative
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COAGULASE TEST
Staphylococcus aureus
Staphylococcus epidermidis
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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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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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CAMP TEST
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
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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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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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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Litmus milk 1 control, 2 pink = acid, 3 white = reduction, 4 & 5 stormy fermentation, 6 blue = alkaline
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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
Substrate
Lactose
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
Lactose
MSA
Mannitol
7.5% NaCl
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Media Tryptic Soy Broth (TSB) MRVP broth MRVP broth Simmons citrate agar
IMViC Product(s) Reagents Indole Kovacs layered on top Organic acids Methyl red Non-acids VP-A & VP-B (Barritts) Na2CO3
Media TSI agar (stab & drag) TSI agar (stab & drag)
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
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
+ 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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Reagent
+ appearance Bubbles Blue ppt. (clumping) Blue color Clearing (lysis of cells)
Test *Alpha
**Beta
Gamma
+ organisms Strep. pneumoniae E.(S) faecalis Staph. epidermidis Strep. pyogenes, E.(S) faecium Staph. aureus
No hemolysis
*Alpha-hemolytic Strep differentiated by optochin sensitivity (S. pneumoniae) **Beta- hemolytic Strep differentiated by bacitracin sensitivity (S. pyogenes)
Substrate Nitrate
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
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Bacterial Infection (Secondary Infection) (sign / symptoms) (Etiologic agent) (Site of Infection)
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)
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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.
DNA-se test
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Enterobacteriaceae
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Gram Stain of Sputum Specimen Showing Capsules Surrounding a Gram Negative Bacillus
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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 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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