Staphylococcus bacteria introduction history techniques to isolate bacteria culture biochemical test antibiotic disc diffusion test

1. ISOLATION OF STAPHYLOCOCCUS
AUREUS BACTERIA FROM
CLINICAL SAMPLES
BY
TUSHA VARSHNEY
BSC BIOTECH 3rd year

2. INTRODUCTION
• The generic name Staphylococcus is derived from the Greek word
“staphyle”, meaning bunch of grapes, and “kokkos”, meaning granule.
The bacteria when seen under a microscope, appear like a branch of
grapes or berries.
• They are gram positive cocci.
• Staphylococcus aureus is a round-shaped bacterium and is frequently
found in the nose, respiratory tract, and on the skin.
• Although S. aureus is not always pathogenic, it is a common cause of skin
infections such as a skin abscess, respiratory infections such as, sinusitis,
and food poisoning.
• S. aureus is non-motile and does not form spores.
• S. aureus reproduces asexually by binary fission.
• Cultural Characteristics: They grow readily on ordinary media within a
temperature range of 10 to 420C, the optimum being 37 0C, and a pH of
7.4-7.6. They are aerobes and facultative anaerobes.

3. HISTORY
• Staphylococcus was first observed in human pyogenic
lesions by Von Recklinghausen in 1871.
• Staphylococcus was then identified in 1884-1929 in
Aberdeen, Scotland, by surgeon Sir Alexander Ogston
in pus from a surgical abscess in a knee joint.
• Anton J. Rosenbach (1842-1923), a German surgeon,
isolated two Strains of staphylococci, which he named
for the pigmented appearance of their colonies:
Staphylococcus aureus, from the Latin aurum for gold,
and Staphylococcus albus (now called epidermidis),
from the Latin albus for white.

4. OBJECTIVE
• The objective of these studies was to identify ways to improve
outcomes of patients with S. aureus infections by investigating
microbial characteristics of S. aureus and treatment options for
patients with S. aureus infections. Additionally, methods to
prevent S. aureus infections from occurring were examined.
• Describe the morphological and physiological characteristics of
bacteria in the genus Staphylococcus.
• List the features by which Staphylococcus aureus and
Staphylococcus epidermidis can be identified.
• Describe the spectrum of diseases caused by Staphylococci.
• Describe the antibiotic assay in body fluids.
• Explain the reasons for performing antibiotic sensitivity tests.

5. MATERIAL REQUIREMENT
• Autoclave
• Hot air oven
• Incubator
• Laminar air flow
• Heating mantle
• Water bath
• Hot plate
• Micropipettes
• Spatula
• Filter paper
• Refrigerator
• Digital weighing machine
• Test tubes
• Glass beakers
• Glass pipettes
• Conical flask
• Glass spreader
• Petri dish
• Measuring cylinder
• Volumetric flask
• Funnel
• Glass rod
• Erlenmeyer flask
• Dropper
• Cotton
• Marker
• Forceps
• Aluminum foils
• Bacteriological loop/straight wire
• Slides
• Red top tube
• Tube stand
• Burner
• Reagents
• H2O2
• Normal saline
• Distilled water
• Plasma
• Paraffin oil
• Mannitol
• Antibiotics

6. METHODS
Laboratory diagnosis:
• The specimens to be collected depend on the type of
lesion (for example, pus from suppurative lesions, sputum
from respiratory infections). In cases of food poisoning,
feces and the remains of suspected food should be
collected. For the detection of carriers, the usual
specimen is the nasal swab. Swabs from the perineum,
pieces of hair and umbilical stump may be necessary in
special situations.
• Diagnosis may readily be made by culture. The specimens
are plated on blood agar and MacConkey’s agar.

7. Composition of MacConkey’s Agar
Ingredients Amount
Peptone (Pancreatic digest of gelatin) 17 gm
Proteose peptone (meat and casein) 3 gm
Lactose monohydrate 10 gm
Bile salts 1.5 gm
Sodium chloride 5 gm
Neutral red 0.03 gm
Crystal Violet 0.001 g
Agar 13.5 gm
Distilled Water Add to make 1 Liter

8. PROCEDURE
• Suspend 49.53 grams of
dehydrated medium in 1000ml
purified/distilled water .
• Heat to boiling to dissolve the
medium completely .
• Sterilize by autoclaving at 15lbs
pressure (1210c) for 15 min or
as per as validated circle.
• Cool to 45-500c.
• Mix well before pouring into
sterile Petri plates.
• The surface of the medium
should be dry when
inoculated.

9. BLOOD AGAR
• Blood agar consists of a base containing a protein
source (e.g. Tryptones), soybean protein digest,
sodium chloride (NaCl), agar and 5% sheep blood.
Composition of Blood Agar:
• Pancreatic digest of casein
• Papaic digest of soy meal
• NaCl
• Agar
• Distilled water
• Combine the ingredients and adjust the pH to 7.3.
Boil to dissolve the agar, and sterilize by autoclaving

10. PROCEDURE
• Prepare the blood agar base as instructed by the
manufacturer. Sterilize by autoclaving at 121°C for
15 minutes.
• Transfer thus prepared blood agar base to a
50°C water bath.
• When the agar base is cooled to 50°C, add sterile
blood agar aseptically and mix well gently. Avoid
formation of air bubbles. You must have warmed
the blood to room temperature at the time of
dispensing to molten agar base.
• Dispense 15 ml amounts to sterile Petri plates
aseptically
• Label the medium with the date of preparation
and give it a batch number (if necessary).
• Store the plates at 2-8°C, preferably in sealed
plastic bags to prevent loss of moisture. The shelf
life of thus prepared blood agar is up to four
weeks.

11. STREAKING METHOD
• The plated are ready for streaking. The most common
method of inoculating an agar plate is streaking.
• With this method, a small amount of sample is placed on
the side of the agar plate either with the swab, or a drop
from an inoculating loop if the sample is a liquid) and a
well is made.
• A sterile loop (flamed until red hot, then cooled by
touching the agar way from the inoculated sample) is then
used to spread the bacteria out in one direction from the
initial site of inoculation. This is done by moving the loop
from side to side, passing through the initial site.
• Now the plates are placed in incubator. Incubation of
culture at 370c is standard practice in the culture of
bacteria pathogenic to man and then the plates. Colonies
appear after overnight incubation.

12. COLONIES

13. Gram’s staining procedure
• First of all take the clean microscope slide. Now put few drops Of normal saline
on the slide.
• With the help of sterile straight wire take the colonies from blood agar and mix in
normal saline.
• The smear is fixed to the slide by gentle heating over a flame.
• Once fixation is complete, crystal violet is applied to cover the smear. The stain is
left on the slide for one minute.
• The crystal violet stain is washed off. Now, the smear is then covered with Gram’s
iodine for one minute.
• The Gram’s iodine is poured off the slide. The slide is then washed with acetone
or 95% alcohol to decolorize the smear. The acetone/alcohol should be poured
on the smear till all the stain runs off.
• The slide is washed with distilled or taps water.
• The secondary stain, either safranine or dilute carbol fuchsin is applied on the
smear. The secondary stain should cover the smear for 30 seconds.
• The secondary stain is washed off the slide by distilled or tap water. The smear is
allowed to dry in air or blotted dry by a filter paper.
• The smear is viewed under the oil immersion objective lens of the light
microscope.

15. Procedure of catalase test
Tube Method
• Pour 1-2 ml of hydrogen
peroxide solution into a
test tube.
• Using a sterile wooden
stick or a glass rod, take
several colonies of the 18
to 24 hours test organism
and immerse in the
hydrogen peroxide
solution.
• Observe for immediate
bubbling.
Slide Method
• Use a loop or sterile
wooden stick to transfer a
small amount of colony
growth in the surface of a
clean, dry glass slide.
• Place a drop of 3% H2O2 in
the glass slide.
• Observe for the evolution
of oxygen bubbles.

17. COAGULASE TEST
• Coagulase test is used to differentiate
Staphylococcus aureus (positive) which produce
the enzyme coagulase, from S. epidermis and S.
saprophyticus (negative) which do not produce
coagulase. I.e. Coagulase Negative
Staphylococcus (CONS).
Principle of Coagulase Test
• Coagulase is an enzyme-like protein and causes
plasma to clot by converting fibrinogen to
fibrin.

18. Procedure and Types of Coagulase Test
Slide Test (to detect bound coagulase)
• Place a drop of physiological
saline on each end of a slide, or
on two separate slides.
• With the loop, straight wire or
wooden stick, emulsify a portion
of the isolated colony in each
drop to make two thick
suspensions.
• Add a drop of human or rabbit
plasma to one of the
suspensions, and mix gently.
• Look for clumping of the
organisms within 10 seconds.
• No plasma is added to the
second suspension to
differentiate any granular
appearance of the organism
from true coagulase clumping.
Tube Test (to detect free coagulase)
• Dilute the plasma 1 in 10 in
physiological saline (mix 0.2 ml of
plasma with 1.8 ml of saline).
• Take 3 small test tubes and label as T
(Test), P (Positive Control) and N
(Negative Control). Test is 18-24 hour
broth culture, Positive control is 18-24
hr S. aureus broth culture and Negative
control is sterile broth.
• Pipette 0.5 ml of the diluted plasma
into each tube.
• Add 5 drops (0.1 ml) of the Test
organisms to the tube labeled “T”, 5
drops of S. aureus culture to the tube
labeled “P” and 5 drops of sterile broth
to the tube labeled “N”.
• 32
• After mixing, incubate the three tubes
at 35-37 Degree Celsius.
• Examine for clotting after 1 hours. If no
clotting has occurred, examine at 30
minutes intervals for up to 6 hours.

20. Mannitol salt agar tube
• This is a selective and differential medium
• It is a selective medium since only organisms
(Staphylococcus aureus)that can grow in the presence
of sodium chloride (salt0 in a concentration of 6.5%
are able to grow on this medium.
• It is a differential medium since Staphylococci can be
differentiated on this medium by their ability to
ferment mannitol. Following incubations, mannitol-
fermenting organisms, typically S. aureus strains,
exhibit a yellow halo surrounding their growth, and
non-fermenting strains do not.

21. Oxidative fermentor tube
• Take two tubes of oxidative fermentor.
• With the help of sterile loop mix colonies in
each tube.
• Now add paraffin oil in one tube so that, it cut
the supply of oxygen and bacteria can also
ferment in absence of oxygen.
• Incubate both the tubes for 24 hours.
• Next day we observe that the colour of both the
tubes changes from green to purple. Its shows
that staphylococcus is a aerobic as well as
anaerobic bacteria.

22. Antibiotic sensitivity tests
• Mueller-Hinton agar (MHA) is the best medium to use for routine susceptibility testing.
Formula for Mueller-Hinton agar per liter of purified water
• Beef extract: 2.0 g
• Acid Hydrolysate of Casein: 17.5 g
• Starch: 1.5 g
• Agar: 17.0 g
Preparation of Mueller-Hinton agar
• Suspend 38 g of medium (or the components listed above) in 1 liter of purified water.
• Mix thoroughly.
• Heat with frequent agitation and boil for 1 minute to completely dissolve the components.
• Autoclave at 121°C for 15 minutes.
• Cool to 45°C
• Pour cooled Mueller Hinton Agar into sterile petri dishes on a level, horizontal surface to
give uniform depth.
• Allow to solidify at room temperature.
• Check prepared Mueller Hinton Agar to ensure the final pH is 7.3 ±1 at 25°C.

23. DISC DIFFUSION TEST
Now, the MHA agar plate is ready for antibiotic disc diffusion test
With the help of swab the normal saline mix with colonies are spread
uniformly over the MHA plate.
Antibiotics for Staphylococcus aureus bacteria are:
•Azithromycin (replaced by nitrofurantain in urine c/s)
•Erythromycin
•Clindamycin
•Penicillin/ampicillin
•Amoxicillin
•Cefoxitin/oxacillin
•COT
•Doxycycline
•Ciprofloxacin/ofloxacin (nitrofloxacin in urine c/s)
•Gentamicin

24. ANTIBIOTICS
• The isolates were similarly inoculated
onto the surfaces of plain Mueller-
Hinton agar plates and Gentamicin
(10µg), Amoxycillin/clavulanate (30µg),
Streptomycin (30µg), Cloxacillin (5µg),
Erythromycin (15µg), Chloramphenicol
(30µg), Cotrimoxazole (25µg),
Tetracycline (30µg), Penicillin (10iu),
Ciprofloxacin (5µg), Ofloxacin (5µg),
Levofloxacin (5µg), Ceftriaxone (30µg)
and Amoxycillin (30µg) discs were
placed and incubated at 37oC for 24hrs.
• The Zones of inhibition were measured
and compared with national committee
for clinical laboratory standards (NCCLS)
guidelines. The isolates that were
resistant to methicillin (<17mm) were
termed Methicillin resistant
Staphylococcus aureus (MRSA) while
those with zone of inhibition as
(≥17mm) were termed susceptible.