Exp. No. 5 Isolation and identification of Bacterial pathogens: Staphylococcus aureus, Streptococcus sp., E.coli, Salmonella, Klebsiella , Pseudomonas, Proteus sp.,
Isolation and identification of Bacterial pathogens: Staphylococcus aureus
Introduction
Members of the genus
Staphylococcus are facultative anaerobic, nonmotile. Gram positive cocci that
usually irregular clusters. Staphylococci are responsible for many human
discases. Staphylococci are normally associated with the skin and mucous
membranes of warm-blooded animals,
Aim
To isolate and identify
Staplylococcus aureus from clinical samples/from mixed/ cultures.
To isolate and identify
Staplylococcus aureus from clinical samples/from mixed/ cultures.
Procedure/ Program
Day 1
Check the purity of the culture by streaking the culture on nutrient agar plate, incubate at 37Cfor 24 hours and note Colony morphology of the culture (Pure culture showed uniform morphology). Perform gram staining to look for gram’s nature. If the culture is gram positive, perform the following tests. Inoculate test organism on blood agar plate and incubate it for 24 hours at 37C aerobically. Place Bacitracin, Novobiocin and Furazolidone discs on different corners of blood agar plate and observe for sensitivity pattern. Carry out catalase test by inserting a stick containing culture on H2O2, solution and observed bubble formation. These tests differentiate Staphylococcus from Micrococcus
Day 2
· Inoculate test organism on the basal medium with the carbohydrate glucose. Perform motility test, oxidase test and mannitol salt agar for the differentiate Staphylococcus aureus.
Day 3
· Inoculated the test organism on Baired parker agar, Mannitol salt agar, to confirm the Staphylococcus aureus and incubate at appropriate temperature for appropriate time duration. Perform coagulase test to confirm pathogenic strain of Staphylococcus aureus.
Result
Gram staining showed
positive cocci in clusters, catalase positive, Beta hemolytic on blood agar,
Oxidase negative, and golden yellow colonies on Mannitol salt agar confirmed as
Staphylococcus aureus.
Introduction
Streptococci are gram
positive cocci chain. They are gram positive cocci arranged in important human
pathogen causing pathogenic infection with a characteristic tendency to spread
as as opposed to staphylococcal infection. Streptococci are divided into
obligate anaerobes. The aerobic and facultative anaerobic forms classified
based on their hemolytic properties. In 1919, Brown recognized three types of
reactions namely: are (1) Alpha (x) hemolytic streptococci which produce greenish
discolouration with partial hemolytic around the colonies. (2) Beta (B)
hemolytic streptococci which produce defined clear colourless zone of
hemolysis. (3) Gamma (Y) non-hemolytic streptococci which produce change on the
medium. The great majority of hemolytic streptococci that produce human
infection belong to group-A streptococci which are known as Streptococcus
pyogenes.
Aim
To isolate and identify the bacterial
pathogen, Streptococcus sps.
Materials and Methods
Sample: Broth
or slant culture
Procedure
Gram
staining:
Take a loopful of
sample and smear well on a slide. Add crystal violet and saffranin mordant and
decolourizing agent. The slide is then viewed under a microscope to study the
morphology of the organism.
Culture
media:
Blood
agar:
To a blood agar plate,
streak the organism and inoculate at 37°C for 24 hours. After inoculation, the
plates were observed for beta hemolysis or other types of hemolysis.
Lipid media:
Lipid media plates were
prepared, sterilized, inoculated and inoculated at 37°C for 24 hours. Following
incubation, the plates were observed.
Biochemical
tests:
Various biochemical
tests like glucose fermentation, lactose fermentation, dextrose fermentation,
sucrose fermentation, hydrogen sulphide production test, nitrate production
test, indole test, Methyl red test, Voges Proskauer test, citrate test, oxidase
test, Urease test, catalase test, gelatin hydrolysis, starch hydrolysis, and
lipid hydrolysis tests were performed and the results were observed.
Novobiocin
Sensitivity Test:
Novobiocin
sensitivity test is with a Muller Hinton plate. The test organism is inoculated
and inoculated at 37°C for 24 hours with application of a 30 micro antibiotic
disc to the surface of Novobiocin agar. Following inoculation, the sensitivity
of an organism to the antibiotic is determined by the Kirby - Bauer method.
Result
The colonies were small
(0.5-1.0 mm), circular, semitransparent, low convex discs with an area of clear
hemolytic zone which is promoted by 10% of CO₂. Virulent strain on fresh isolation
from lesions produces a growth. In lipid
media such as glucose or serum, growth occurs a granular turbidity with a
powdery deposit. No pellicle is formed. The organism tests positive for indole
test. From the above obtained results, it can be concluded that the isolated
organism was Streptococcus sps.
Introduction
E. coli and other facultative anaerobes constitute about 0.1% of gut microbiota, and fecal oral transmission is the major route through which pathogenic strains of the bacterium cause disease. Cells are able to survive outside the body for a limited amount of time, which makes them potential indicator organisms to test environmental samples for fecal contamination. A growing body of research, though, has examined environmentally persistent E. coli which can survive for many days and grow outside a host.
The bacterium can be grown and cultured easily and inexpensively in a laboratory setting and has been intensively investigated for over 60 years. E. coli is a chemoheterotroph whose chemically defined medium must include a source of carbon and energy. E. coli is the most widely studied prokaryotic model organism, and an important species in the fields of biotechnology and microbiology, where it has served as the host organism for the majority of work with recombinant DNA. Under favorable conditions, it takes as little as 20 minutes to reproduce.
Aim
To identify
and isolate E.coli.
Materials and Methods
EMB agar,
sample containing culture.
Procedure
E. coli rapidly ferments lactose and is
indistinguishable from most other E. coli on traditional
lactose-containing media. However, unlike approximately 80% of other E. coli,
nearly all isolates of E. coli ferment D-sorbitol slowly, or not at all.
Sorbitol - Mac Conkey (SMAC) agar was developed to take advantage of this
characteristic by substituting the carbohydrate sorbitol for lactose in Mac Conkey
agar and is the medium of choice for isolation of E. coli.
Inoculate stool specimens onto SMAC and
incubate 18-24 hours at 35-37C. Sorbitol-negative colonies will appear
colorless on SMAC. Test sorbitol- negative colonies selected from SMAC with E.
coli antiserum or latex reagents according to the procedures recommended by the
manufacturer. If using latex reagents, it is important to test isolates in the
control latex to detect nonspecific agglutination of organisms with latex.
Manufacturers of latex reagents recommend heating strains that agglutinate in
the latex control reagent and then retesting them in both the antibody-coated
and control latex reagents. However, E. coli have not been shown to
agglutinate in both the antibody-coated and control latex reagents.
Colonies may be tested with antisera
directly from the plate, or subcultured to another nonselective medium (blood
agar, for example) and tested the next day. If colonies are tested directly
from the plate, positive colonies should also be transferred to another medium
for subsequent testing. Although it is more labor-intensive and delays results
by a day, sub-culturing to another medium and testing the next day offers the
advantage of providing more bacterial growth on which to perform the
agglutination assay. The extra growth makes it easier to observe agglutination
and allows repeat testing of the isolate. if necessary. Once one colony from a
plate as been identified as positive, no further colonies from the same plate
need to be tested.
Isolates agglutinating in antiserum or
latex reagent should be identified biochemically as E. coli, since
strains of several species cross-react with antiserum However, because
biochemical confirmation may take 24 hours or longer, an oral report of
presumptive E. coli may be given before biochemical identification is
completed.
Specimens from which sorbitol-negative
colonies have been isolated that agglutinate in antiserum or latex reagent, and
are biochemically E. coli, may be reported as presumptively positive for
E. coli. A preliminary written report should be issued to the clinician
and to public health authorities. It may be useful to note on the laboratory
report that E. coli is an enteric pathogen and can cause non-bloody
diarrhea, bloody diarrhea, and HUS.
Based on the above observation on EMB agar, greenish metallic sheen
colonies are confirmed as E.coli.
Introduction
Enteric fever is a
collective term used for invasive infections caused by a small group of
bacteria called Salmonella. Salmonella are a group of enteric invasive
bacteria. They are responsible for variety of gastrointestinal Infections.
Aim
To identify Salmonella sp. From the
given culture.
Materials and Methods
Sample :Broth or slant culture. Gram staining reagent, Mac
Conkey agar, XLD agar, SS agar, Test
media and reagents, Glass slides, petri plates, test tubes etc.
Procedure/ Program
Day
1
·
Subject the isolated pure culture to
simple stain, gram stain and catalase to know nature of the family of the
isolate.
·
Streak the isolate on Mac Conkey agar
and incubate at 37C i0r 24 hours.
·
Subject the isolate to Motility test,
Indole test and TSI test. It is helpful to understand the probable nature of
the isolate.
Day
II
·
Perform MRVP, Urease, Nitrate,
Deaminease, Decarboxylase, Melonate, KCN and carbohydrate fermentation etc.
Day III
·
Confirm probably identified bacterial
isolate by streaking it on, XLD agar, SS agar. Incubated all the media at 37 C
for 24 hours and observed the colony morphology.
Result
Based on the growth on selective
medium, it was confirmed as Salmonella sp.
Introduction
Klebsiella pneumoniae
is an important opportunistic pathogen, which was reported worldwide. It can
infections of respiratory tract, nasal mucosa and pharynx and generally results
in primary pneumonia. Klebsiella pneumoniae is facultative anaerobic,
non-motile, rod shaped gram-negative bacteria with a prominent polysaccharide
capsule. This capsule causes the entire cell surface, accounts for the large
appearance of the organism on gram stain and provides resistance against many
host defense mechanisms.
Members of the Klebsiella genus
typically express types of antigens on their cell surface. The first is a lipopolysaccharide
(0 antigen); the other is a capsular polysaccharide (k antigen). Both of these
antigens contribute to pathogenicity. About 77 k antigens and one o antigen
exists. The structural variability of these antigens forms the basis of
classification into various serotypes. The virulence of all serotypes appear to
be similar. Capsule plays a very important role in virulence so we study with
the aim to utilize capsules as an antigen to develop simple and rapid
diagnostic method which can be utilized anywhere without the use of any
sophisticated equipments specially for field use as a preliminary routine test
for detection of Klebsiella pneumoniae.
Aim
To isolate and identify the bacterial
pathogen, Klebsiella pneumoniae.
Materials and Methods
Klebsiella cultures
were characterized by number of biochemical reaction such as TSI test,
carbohydrate fermentation of glucose, lactose, sucrose and mannitol, IMVic test
and urease test. The motility test was studied using SIM medium (Sulphide
Indole Motility) or hanging drop method.
Procedure
Klebsiella infections are typically diagnosed
with a lab test that examines a sample of the infected tissue, such as
blood, urine, or sputum (a mixture of saliva and mucus). Imaging tests, such as
ultrasounds, X-rays, and CT scans, may also help your doctor with the
diagnosis. Klebsiella pneumoniae produces large dome shape, mucoid colony on
BHI agar and Lactose fermenting pink colony on Mac Conkey agar. It confirm by
biochemical test like indole negative, MR negative, VP positive, Citrate
positive, Oxidase negative and catalase positive.
Result
Gram
staining:
Gram negative rods were seen under oil
immersion objective.
Biochemical
tests:
Non-motile form was obtained producing
acid slant and acid bult and gas production on Ts production on TSI slant. The
fermentation of sugars (glucose, sucrose, lactose and mannitol) was obtained
with acid and gas production. It was found to be negative for indole
production, methyl red test, urease test and H2S test.
Colony
morphology:
Large dome shaped mucoid
sticky colonies were nutrient agar medium. Bright pink colonies were on
observed due to lactose fermentation on
MacConkey agar. From the above obtained results, it can be concluded that the
isolated organism was Klebsiella pneumoniae.
Introduction
Pseudomonas aeruginosa is
an opportunistic pathogen of humans. It is a Gram-negative, aerobic
rodbelonging to the family Pseudomonadaceae. These bacteria are common
inhabitants of soil and water. Almost all strains are motile by means of a
single polar flagellum. Infection associated with Pseudomonas aeruginosa
are Endocarditis, Pneumonia, Bacteremia, Septicemia, meningitis and brain
abscesses, otitis, keratitis, neonatal ophthalmia,Osteomyelitis, Urinary tract
infections. Gastrointestinal infections like pediatric diarrhoea, typical
gastroenteritis, and necrotizing enterocolitis, skin infections like burns,
trauma or dermatitis; folliculitis, acne vulgaris like symptoms.
Aim
To identity Pseudomonas aeruginosa
from the given culture.
Materials and Methods
Sample: Broth or stab
or slant culture.
Chemicals and Media
Required:
Gram staining reagent,
MacConkey agar, Cetrimide agar, Biochemical test media & reagents, Glass
slides, petri plates, test tubes etc.
Procedure
Day 1
Checked the purity of the
culture by streaking the culture on nutrient agar plate, incubated at 37°Cfor
24 hours and note Colony morphology of the culture (Pure culture showed uniform
morphology)
Perform gram staining
to look for gram’s nature and catalase and oxidase test. Inoculated test
organism on MacConkey agar plate and incubated it for 24 hours at
37Caerobically and performed Motility test, Indole test and TSI test. It is
helpful to understand the probable nature of the isolate and these will
differentiateEnterobacteriaceae from other aerobic family of human pathogens.
Day 2
Perform MRVP, Urease,
Nitrate, Deaminease, Decarboxylase, Melonate, KCN and carbohydrate fermentation.
Tests along with EMB media inoculation to confirm generic level identification
of the isolate.
Day 3
Probably Identified
bacterial isolate is confirmed by streaking it on Cetrimide agar and incubate
at 37Cfor 24hours and observed the colony morphology.
Result
From the above observation, it was
confirmed as Pseudomonas aeruginosa.
Introduction
Proteus are
opportunistic pathogen, strictly do not belong to the group of coliform
bacilli. "The name "Proteus" refers to the pleomorph, after the
Greek god "Proteus" - who could assume different shapes. Proteus
sps found in intestine of human and animal in soil, sewage and water. Proteus
mirabilis causes abdominal and wound infections, septicemia and
occasionally meningitis and chest infection. Proteus vulgaris is
occasionally isolated from urine, pus and other specimens.
Aim
To isolate and identify the bacterial
pathogen, Proteus sps.
Materials and Methods
Procedure
Gram
staining:
Take a loopful of
culture and smear well on a slide. Add crystal violet and safranin with mordant
and decolourizing agent. The slide is then viewed under a microscope to study
the morphology of the organism.
Culture
media:
Different
culture media namely,
(1)
Nutrient agar
(2)
Mac Conkey agar
(3)
Blood agar
(4)
SS agar
were prepared, sterilized, inoculated
and incubated at 37°C for 24 hours. After incubation, the plates were observed.
Biochemical
test:
Various biochemical
tests like glucose fermentation, lactose fermentation, dextrose fermentation,
sucrose fermentation, hydrogen sulphide production test, nitrate reduction
test, Methyl red test, Voges Proskauer test, indole test, citrate test urease
test, oxidase test, catalase test, gelatin hydrolysis, starch hydrolysis and
lipid hydrolysis teste were performed and the results were observed.
Novobiocin
sensitivity test:
Novobiocin
sensitivity test is with a Muller - Hinton Agar plate. The test organism is
inoculated and incubated at 37°C for 24 hours with the application of a 30 microgram
of Novobiocin antibiotic disc to the surface of agar. Following incubation, the
sensitivity of the organism to the antibiotic is determined by the Kirby-Bauer method.
Result
Proteus sps.
are gram negative rods. They non-lactose fermenting and produce translucent
colonies on Mac Conkey agar. They appear as swarming growth agar. Black
colonies are seen on ss on blood P. mirabilis is indole agar. negative
and P. vulgaris is indole positive. From the above obtained results, it
can be concluded that the isolated organism is Proteus sps.
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