This document discusses the laboratory diagnosis of Staphylococcus, including sample collection, direct smear microscopy, culture techniques, biochemical reactions, antibiotic sensitivity testing, and typing of Staphylococcus aureus. Appropriate samples are collected based on the site of infection and transported to the laboratory for analysis. Direct smear microscopy can identify Gram-positive cocci clusters. Culture techniques help isolate and identify Staphylococcus colonies based on morphology and biochemical reactions provide further characterization. Antibiotic sensitivity testing determines effective treatment options and typing methods like bacteriophage typing are used for epidemiological purposes.

1. LABORATORY DIAGNOSIS
OF STAPHYLOCOCCUS
MADE BY:
SHALINI BISHT
Saturday, February 18, 2017 1

2. LABORATORY DIAGNOSIS
 Sample collection and Transportation
 Direct smear Microscopy
 Culture
 Biochemicals
 Typing of Staphylococcus aureus
 Antibiotic Sensitivity Testing (AST)
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3. SAMPLE COLLECTION
 Type of sample depends on the site of
infection.
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Infection Specimen
Suppurative lesion Pus, wound swab
Respiratory infection Sputum
UTI Mid stream urine
PUO, Bacteremia Blood
Food poisoning Feces, Vomitus, food
Carriers Nasal and perianal swab

4. DIRECT SMEAR MICROSCOPY
 Staphylococcus appear as GPC
measuring 0.5-1.5 microns
 Occur singly, in pairs, short chains or
clusters
 Present within and outside PMNs
 Reporting of direct smears:
◦ quantitation of cell types and
microorganisms
◦ Eg. Many pus cells along with moderate
number of GPC seen
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6. CULTURE
• Specimens are inoculated onto the suitable media.
 Plates incubated for 18-24 hour at 37°C.
 On nutrient agar
 Colonies are golden yellow and opaque with smooth
glistening surface, 2-4 mm in diameter, circular, convex,
shiny & easily emusifiable.
(Most strains produce non diffusible Golden yellow
pigment)
 Nutrient Agar slope
◦ Confluent growth, Oil paint appearance.
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7.  Blood agar
◦ Colonies similar to those on Nutrient Agar
◦ Colonies are beta-hemolytic
 Liquid medium
◦ Uniform turbidity
 MacConkey Agar
◦ Small pink colonies due to Lactose
fermentation
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9.  Selective media
 MSA – 1% Mannitol + 7.5% NaCl +
phenol red
 Salt milk agar – 6.5% NaCl + 10%
skimmed milk
 Ludlam’s medium – Lithium chloride
and tellurite
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10. Saturday, February 18, 2017 10

11. BIOCHEMICAL REACTIONS
•Catalase : positive
•Coagulase test : positive
•Oxidase : negative
(Except S.sciuri group i.e., S.sciuri, S.lentus,
S.vitulinus)
•Ferment glucose, lactose, maltose, sucrose and
mannitol, with production of acid but no gas
•Indole : negative
•MR test : positive
•VP test : positive
•Gelatin liquefaction : positive
•Phosphatase : positiveSaturday, February 18, 2017 11

12. Catalase test
•Done to distinguish staphylococci from
streptococci (catalase negative)
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13. Coagulase test
•Done to distinguish pathogenic strain (S.aureus)
from non-pathogenic strains.
•2 Methods of coagulase detection are:
(1) Slide coagulase test : detects bound
coagulase
(2) Tube cogulase test : detects free coagulase
 (other coagulase +ive staphylococci are
S.intermedius, S.hyicus)
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15. Gelatin liquefaction
Principle: this test is used to determine the ability
of an organism to produce proteolytic enzyme
(gelatinase) that liquefies the gelatin.
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16. Methyl Red test
Principle: this test detects the production of
sufficient acid during fermentation of glucose by
bacteria and sustained maintenance of ph below
4.5
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17. Voges-Proskauer (VP) test
Principle: the test depends upon the production
of acetoin from pyruvic acid in the media. In the
presence of alkali & atmospheric oxygen, acetoin
is oxidised to diacetyl which reacts with alpha
naphthol to give red color.
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18. DNA Hydrolysis
•Principle : this test is used to determine the ability of
an organism to hydrolyze DNA. Green color of the
medium is due to DNA-methyl green complex. If the
organism growing on the medium hydrolyzes DNA,
the green color fades & the colony is surrounded by
a colorless zone.
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19. Phosphatase test
Principle: Staphylococci are grown on nutrient agar
containing sodium phenolphthalein diphosphate and
incubated overnight at 37°C. The plate is exposed to
ammonia vapours. The pink color of the colonies
indicate a positive result.
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20. S.aureus v/s CONS
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21. BACTERIOPHAGE TYPING
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22.  Epidemiological purpose to trace
source of infection.
 Useful in outbreaks like food poisoning
in a community.
 Typing methods:
◦ Phenotypic-bacteriophage typing:
staphylococci are typed based on their to
bacteriophages.
◦ Molecular typing: DNA finger-printing,
ribotyping, PFGE etc.
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23. Bacteriophage typing
 Method
◦ Test strain inoculated as lawn culture on NA.
◦ Drops of routine test dose of known set of
different phages are spot inoculated &
incubated.
◦ Zone of lysis will be produced in those areas
where test strain is susceptible to phages
applied.
◦ If strain lysed by phages 29, 52A, 79, but not
other phages; it is designated as phage type
29/52A/79
◦ National Reference Centre: MAMC, NewSaturday, February 18, 2017 23

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25. ANTIBIOTIC SENSITIVITY TESTING
This is important as staphylococci develop
resistance to drugs readily.
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26. Why AST has become a
necessity ??
Bacteria have the ability to develop resistance
following repeated or subclinical doses, so more
advanced antibiotics are required to overcome
them.
 Antibiotic sensitivity test: A laboratory test
which determines how effective antibiotic
therapy is against a bacterial infections.
 Testing will assist the clinicians in the choice of
drugs for the treatment of infections.
 Helps in the local pattern of antibiotic
prescribing. Saturday, February 18, 2017 26

27. Methods of AST
•Performed on MHA by Kirby-Bauer Disc diffusion
method.
•Following antibiotics are employed for
staphylococcus:
•Amoxyclav
•Clindamycin
•Cefoxitin
•Ciprofloxacin
•Erythromycin
•Gentamicin
•Linezolid
•Levofloxacin
•Penicilin-G
•Vancomycin
•Teicoplanin Saturday, February 18, 2017 27

28. Kirby-Bauer method
1. Dry the agar plates (MHA) & label them.
2. Dip a sterile swab into the broth and express
any excess moisture by pressing the swab
against the side of the tube.
3. Swab is streaked as a lawn (lawn culture)
onto a Mueller-Hinton agar (in 3 directions
to ensure confluence).
4. The anitibiotic(s) disk will be placed onto the
MHA plate.
5. The plate is incubated and is examined for
resistance and sensitivity pattern the
following day.
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29. MRSA
•Methicillin-resistant S. aureus.
•First reported in 1960s.
•May colonize mucosal or epithelial surfaces,
(common : anterior nares)
•Nosocomial pathogen.
•Shows Resistant to penicillins,
cephalosporins, carbapenems,
monobactams.
•Vancomycin resistance is rare – so far
•Hospital-acquired (HA MRSA)
•Community-acquired cases now (CA MRSA)
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30. Predisposing factors for
MRSA
•Prolonged & repeated hospitalization
•Indiscriminate use of antibiotics
•Intravenous drug abuse
•Presence of indwelling medical devices
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31. MECHANISM
•MRSA contains the mecA gene which is responsible
for the production of an altered plasma (cell)
membrane-bound enzyme, penicillin-binding protein
2a (PBP- 2a.)
•The altered PBP 2a while able to perform its cell-wall
synthesis function, has a lower affinity and does not
bind to beta-lactam antibiotics
•Thus, the presence of the mecA gene confers
resistance to all beta-lactam antibiotics such as
methicillin.
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32. • Vancomycin remains the drug of choice for
treatment of infections caused by MRSA,
although it is intrinsically less active than the
antistaphylococcal penicillins.
•Combinations of vancomycin with ss-lactam
antibiotics may be synergistic in vivo against
MRSA strains, including those with intermediate
susceptibility to vancomycin.
•Given the increasing prevalence of MRSA in
hospitals and in community settings, alternative
approaches are needed for treatment of
infections caused by MRSA.Saturday, February 18, 2017 32

33. Preventive measures
•Isolation & treatment of MRSA patients.
•Detection of carriers among hospital staff, their
isolation & treatment.
•Avoid indiscriminate usage of antibiotics.
•Following strict aseptic technique
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34. •HAND WASHING STILL CONTINUES TO REDUCE
SEVERAL INCIDENCES OF MRSA SPREAD IN
HEALTH CARE
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35. Detection of MRSA
•MRSA is determined by disc diffusion test using
cefoxitin (30µg) disc on MHA with 2% NaCl & 104
cfu/ml inoculum and incubated at 33-35°C for 24
hour.
•As per CLSI guidelines inhibition zone of </= 21
mm was taken to be MRSA.
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36. Coagulase Negative
Staphylococci
Two species of coagulase negative
Staphylococci can cause human infections:
1. Staphylococcus epidermidis
2. Staphylococcus saprophyticus
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37. S. epidermidis:
•It is a common cause of stitch abscesses.
•It has predilection for growth on implanted
foreign bodies such as artificial valves, shunts,
intravascular catheters and prosthetic appliances
leading to bacteremia.
•In persons with structural abnormalities of
urinary tract, it can cause cystitis.
•Endocarditis may be caused, particularly in drug
addicts.
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38. S.saprophyticus:
•It causes urinary tract infections, mostly in
sexually active young women.
•The infection is symptomatic and may involve
the upper urinary tract also.
•Men are infected much less often.
•It is one of the few frequently isolated CoNS that
is resistant to Novobiocin
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39. Other coagulase negative staphylococci:
•S.haemolyticus
•S.saprophyticus
•S.warneri,
•S.hominis,
•S.epidermidis
•S.caprae
•S.lugdunensis
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