Bacis of microbiology - Part 1

1. MICROBIOLOGY

2. MICROBIOLOGY
Study of micro organisms
E g. Bacteria , Virus, Fungi, Protozoa

3. Sizes of Microbes
Virus – 10 to 1000 nanometers
Bacteria – 0.1 to 5 micrometers
Human eye can see .1 mm

4. Bacteria can be differentiate by stain called Gram Stain

6. 
GRAM STAINING PROCEDURE

Prepare a heat fixed smear of the culture
you wish to examine

Flood the smear with crystal violet (30 sec.
to 2 min)

Quickly and gently wash off excess stain (2
seconds)

Flood the smear with Grams iodine (1
minute)

Decolorize with alcohol (10-20 seconds or
until the excess alcohol which flow off the
slide is colorless)

Quickly and gently wash off excess stain (2
seconds)

Flood the smear with safranin (carbolfuchsin)
(30 sec to 2 min.)

Quickly and gently wash off excess stain (2
seconds)

Blot dry with bibulous paper

Examine your slide under the microscope.
Record sketches of the organisms, size,
color, morphology, and culture identification.

7. Bacteria (Micro organisms)
Round or spherical shape Rod or comma shape
Cocci bacilli
Gram positive cocci and Gram negative bacilli and
Gram negative cocci Gram positive bacilli

8. 
G+ G-

10. STAPHYLOCOCCUS DISEASES
Staphylococcal infections are among the most common of bacterial infections and range
From the trivial to the fatal, characteristically localised pyrogenic lesions, in contrast to the
Spreading nature of streptococcal infections common staphylococcal infections are following
Skin and Soft tissue (abcess, breast abcess, wound, burns, cellulitis)
Musculoskeletal : Osteomyelitis, arthritis,
Respiratory: Tonsillitis, Pharyngitis, Sinusitis, otitis, bronchopneumonia, lung abscess,
Rare pneumonia.
Central nervous system: Abscess, meningitis,
Endovascular : Bacteraemia, Septicaemia, Endocarditis
Urinary : Staphylococci are uncommon in routine urinary tract infections, through they do
Cause infection in association with local instrumentation, implant or diabetes, urinary isolated
staphylococci are to be significant even with low colony count, as they are related to
Bacteraemia.

11. Toxigenicity
Amongst the more common toxins secreted by S. aureus are exfoliative toxin,
enterotoxin, and toxic-shock syndrome toxin-1 (TSST-1).
exfoliative toxin
The exfoliative toxins of Staphylococcus aureus are responsible for the
staphylococcal scalded skin syndrome, a blistering skin disorder that particularly
affects infants and young children, as well as adults with underlying disease.
Enterotoxins
Enterotoxins are frequently cytotoxic and kill cells by altering the apical membrane
permeability of the mucosal (epithelial) cells of the intestinal wall. They are mostly
pore-forming toxins (mostly chloride pores), secreted by bacteria, that assemble to
form pores in cell membranes. This causes the cells to die.
Foods that are associated with staph food poisoning include:
Meats,Poultry and egg products,Salads such as egg, tuna, chicken, potato, and
macaroni,Bakery products such as cream-filled pastries, cream pies, and chocolate
eclairs,Sandwich fillings,Milk and dairy products.
Toxic-shock syndrome toxin-1 (TSST-1)
Menstrual toxic shock syndrome (mTSS) is thought to be associated with
colonization with toxic shock syndrome toxin 1 (TSST-1)-producing Staphylococcus
aureus in women, It causes toxic shock syndrome (TSS) by stimulating the release

12. An aerobic organism or aerobe is an organism that can survive and grow in an
oxygenated environment
An anaerobic organism or anaerobe is any organism that does not require molecular
oxygen for growth. It may react negatively or even die if free oxygen is present.
facultative aerobe one that can live in the presence of oxygen, but does not require it
Facultative anaerobes are bacteria that can grow in both the presence or absence
of oxygen
obligate aerobe one that cannot live without oxygen.

14. Blood agar

15. Mac Conkey agar

16. Mac conkey agar
Composition of MacConkey 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
The selective action
of this medium is attributed to crystal violet and bile salts, which are inhibitory to most
species of gram-positive bacteria.
In MacConkey agar without Crystal violet and bile salts
Staphylococcus spp Lactose fermenter Small pink colonies, 1-2mm in diameter,
opaque
Enterococcus spp Lactose fermenter Dark pink to red, very minute, translucent

17. Culture characteristics
* They grow readily in ordinary media within a temperature of 10- 42 degree celcius
* The optimum temperature is 37 degree celcius
* pH – 7.4 to 7.6
* Pigment production occurs optimally at 22 degree celcius and only on aerobic
culture

18. Beta-hemolysin breaks down the red blood cells and hemoglobin completely.
This leaves a clear zone around the bacterial growth. ...
Alpha-hemolysin partially breaks down the red blood cells and leaves a
greenish color behind. This is referred to as α-hemolysis (alpha hemolysis).
Gamma hemolysis is a lack of hemolysis in the area around a bacterial colony.
A blood agar plate displaying gamma hemolysis actually appears brownish.
This is a normal reaction of the blood to the growth conditions used
(37° C in the presence of carbon dioxide).
BAP tests the ability of an organism to produce hemolysins, enzymes that
damage/lyse red blood cells (erythrocytes). The degree of hemolysis by these
hemolysins is helpful in differentiating members of the genera Staphylococcus,
Streptococcus and Enterococcus. Beta-hemolysis is complete hemolysis
HEMOLYSIS

19. Beta Hemolysis

20. Alpha Hemolysis

21. Gamma Hemolysis

22. CATALASE TEST
* The catalase test is a particularly important test used to determine whether a
gram-positive cocci is a staphylococci or a streptococci. Catalase is an enzyme
that converts hydrogen peroxide to water and oxygen gas. The test is easy to
perform; bacteria are simply mixed with H2O2.
* The enzyme neutralizes the bactericidal effects of hydrogen peroxide, and its
concentration in bacteria has been correlated with the pathogenicity of the
Organism.
* Under the aerobic condition, 3% H2O2 is used,
whereas 15% H2O2 is used under anaerobic conditions.
* The catalase test has been extensively used over the years as it allows the
differentiation of catalase-positive organisms like staphylococci from

23. Principle of Catalase Test
* The metabolic activity of aerobic and facultative anaerobic microorganisms produce
toxic by-products like hydrogen peroxide and superoxide radical (O2—).
* These products are toxic to the organisms and might even result in cell lysis if not
broken down. In the case of pathogenic organisms, different mechanisms are found that
break down these products to non-toxic substances.
* Bacteria capable of synthesizing the enzyme catalase hydrolyze hydrogen peroxide into
water and gaseous oxygen, which results in the liberation of gas bubbles.
H2O2 ———–> H2O + O2
catalase
* The production of catalase thus protects the organism against the lethal effect of
hydrogen peroxide accumulated at the end of the aerobic metabolism.
* The presence of the catalase enzyme can be demonstrated by adding hydrogen
peroxide to the bacterial inoculum, which results in the rapid liberation of oxygen bubbles.
The lack of enzyme is demonstrated by the absence of such bubbles.

24. Microorganism Tested
* Young (18 hours old or less, if possible) colonies of bacteria growing on agar media,
preferably Blood Agar.
* For anaerobes, the colonies should be exposed to air for 30 minutes prior to testing.
Materials required:
Hydrogen peroxide reagent
30% H2O2 for Neisseria
15% H2O2 for anaerobes
3% H2O2 for other bacteria (purchase or dilute 30% 1:10 in deionized water prior to
use)Supplies
Glass slide
Sterile wooden or glass sticks or platinum loops or wires

25. Procedure of Catalase Test
1. Slide Method
A microscope slide is placed inside a petri dish. The use of a petri dish is optional and is
used to limit catalase aerosols, which might carry viable bacterial cells.
A small amount of organism is collected from a well-isolated 18- to 24-hour colony with a
sterile inoculating loop or wooden applicator stick and placed onto the microscope slide.
However, no agar must be picked up with the colony, especially when the culture is
picked up from blood agar.
A drop of 3% H2O2 onto the organism on the microscope slide by using a dropper or
Pasteur pipette.
The formation of bubbles is observed against a dark background to enhance readability.

26. 2. Tube Method
* About 4 to 5 drops of 3% H2O2 are added to a test tube.
* Using a wooden applicator stick, a small amount of organisms from a well-isolated 18-
to 24-hour colony is collected and placed into the test tube.
*The tube is placed against a dark background and observed for immediate bubbles.

27. Quality Control
* As a form of quality control, the following organisms can be used for positive and
negative results:
* Staphylococcus aureus: Catalase positive.( ATCC Strains)
* Streptococcus pyogenes: Catalase-negative. ( ATCC Strains)

28. Result and Interpretation of Catalase Test
* The positive test is demonstrated by the immediate appearance of
bubbles.
* The appearance of one or two bubbles represents a weak reaction.
A negative test is represented by no bubbles or a few bubbles after 20 s.

29. Uses of Catalase Test
* Catalase test is essential for differentiating catalase-positive Micrococcaceae and
Staphylococcaceae from catalase-negative Streptococcaceae.
* The test also allows differentiating aerobic and obligate anaerobic organisms.
*The value of the test has been found in the presumptive differentiation among certain
Enterobacteriaceae.
* This is an important test for the differentiation of aerotolerant strains of Clostridium,
which are catalase-negative, from Bacillus, which are catalase positive.
* This test has been used to differentiate between genera and is also valuable in the
speciation of certain gram-positive organisms such as Aerococcus

30. Limitations of Catalase Test
* RBCs contain catalase, and thus, in order to avoid false-positive results, blood agar
should not be picked up with the colony. If a colony is difficult to pick up or doesn’t grow
well, the test can be repeated from the culture on a different media.
* The test should not be tested from Mueller-Hinton agar.
* Collecting colonies with metal bacteriological loop materials might yield false-positive
results; however, platinum loops do not yield false-positive results.
* Because the enzyme is present in viable cells only, colonies that are older than 24
hours should not be used. Older cultures may give false-negative results.
* Reversing the order of adding the reagent to the colony might result in false-negative
results.
* The reagent and the colony should not be mixed.
* Some strains of S. aureus may appear catalase-negative by drop method so the test
should be repeated with the tube method.
* 30% H2O2 is extremely caustic to the skin. If contact occurs, wash immediately with
70% ethyl alcohol, not water.

31. Modified oxidase Test
Microdase Disk is a reagent-impregnated disk recommended for use in qualitative
procedures to aid in the differentiation of Staphylococcus from Micrococcus by the
detection of the oxidase enzyme.
The oxidase method was originally described by Kovacs in 1956 as a method of
differentiating gram-negative bacilli. However, in 1981, Faller and Schleifer modified
Kovacs’ oxidase reagent by utilizing a tetramethyl-pphenylenediamine (TMPD) in
dimethyl sulfoxide (DMSO).This is referred to as the modified oxidase test.

32. Principle
The test is a rapid method to differentiate Staphylococcus from
Micrococcus spp. by detection of the enzyme oxidase. For the detection of
oxidase enzyme a filter paper circular disks impregnated with tetramethyl-p
-phenylenediamine dihydrochloride (oxidase reagent) in dimethyl sulfoxide
(DMSO) are used. The modified oxidase reagent is prepared as 1% (w/v)
tetramethyl-p-phenylenediamine in certified grade dimethyl sulfoxide. DMSO
provides solubility and stability against auto-oxidation and also aids in the
permeability of cells to the reagent. In the presence of atmospheric oxygen, the
oxidase enzyme reacts with the oxidase reagent and cytochrome C to form the
colored compound, indophenol indicated as blue or purplish blue coloration on the
disc after the introduction of bacterial colony on the disc.
All micrococci possess cytochrome c, whereas most staphylococci, with few
exceptions, lack this type of cytochrome. The oxidase reagent substantiates the

33. Oxidase Disc:
Filter paper disks impregnated with tetramethyl-p-phenylenediamine dihydrochloride
(oxidase reagent) in dimethyl sulfoxide (DMSO).
Method
Testing should be performed on aerobic, catalase-positive, gram-positive cocci.
* Using forceps, place the disk in an empty petri dish or on a clean glass slide.
* Using a wooden applicator stick, rub a small amount of several colonies of an 18- to 24
-hour pure culture grown on blood agar onto a small area of the microdase disk.
Note: Do not rehydrate the disk before use.
* Incubate at room temperature for 2 minutes.
* Examine for a blue color development.

34. Expected results
Positive: Development of blue to purple-blue color within 2 minutes time
Negative: No color change (white to gray color remains).
Uses
This test is used to differentiate Micrococci from Staphylococci. (Micrococci should yield
a positive result. Staphylococci should yield a negative result, with the exception of
Staphylococcus sciuri, S. lentus and S. vitulinus.

35. Limitations
* Staphylococci should yield a negative color change, except for S. sciuri, S. lentus, and
S. Vitulus.
* Bacteria from a culture grown on Blood agar for 24 – 36 hours must be used. Cultures
which are too young or too old may give inaccurate results.
* Macrococcus species and Kocuria kristinae are also known to possess cytochrome
* Microdase is not designed for routine testing for oxidase activity in organisms other
than Staphylococcus and Micrococcus.

36. Coagulase Test:
Coagulase test is used to differentiate Staphylococcus aureus (positive) from
coagulase-negative staphylococci (CoNS). Coagulase is an enzyme produced by S.
aureus that converts soluble fibrinogen in plasma to insoluble fibrin. Staphylococcus
aureus produces two forms of coagulase, bound and free.
* Slide coagulase test is done to detect bound coagulase or clumping factor.
* Tube coagulase test is done to detect free coagulase.

37. Slide Coagulase Test
Slide coagulase test is done to detect “bound coagulase” or “clumping factor”. Clumping
factor is a fibrinogen binding cell surface receptor present in cell wall of most, but not all,
S. aureus. Clumping factor acts directly on the fibrinogen in the plasma resulting in
clumping. Slide coagulase test is rapid, but it requires several colonies and lacks
sensitivity.
Slide coagulase test
As the clumping factors are not present in all S. aureus, some strains will give a negative
slide coagulase test. In addition, clumping factor can be masked by cell surface
capsular polysaccharides. So, the negative slide tests must be confirmed by tube
coagulase test. Other species of Staphylococcus, namely, S. lugdunensis and S.
schleiferi may give positive slide coagulase test results.

38. Slide Coagulase Test Procedure
* Emulsify a staphylococcal colony in a drop of water on a clean and grease-free glass
slide with a minimum of spreading (If the isolate does not form a smooth, milky
suspension, do not proceed with the test).
* Make similar suspensions of control positive and negative strains to confirm the proper
reactivity of the plasma.
* Dip a flamed and cooled straight inoculating wire into the undiluted plasma at room
temperature, withdraw, and stir the adhering traces of plasma (not a loopful) into the
staphylococcal suspension on the slide. Flame the wire and repeat for the control
Suspensions.
* Read as positive a coarse clumping of cocci visible to the naked eye within 10 seconds.
* Read as negative the absence of clumping or any reaction taking more than
10 seconds to develop, but re-examine any slow reacting strains by the tube coagulase test.

39. Observation
Coagulase positive: Macroscopic clumping in 10 seconds or less in coagulated
plasma drop and no clumping in saline or water drop.
Coagulase-negative: No clumping in either drop
Interpretation
Slide coagulase test is the main method used to identify S. aureus in clinical
laboratories but it has some limitations.
* About 15% of ordinary strains of S. aureus and many more of MRSA give negative
reactions.
* Few species of coagulase-negative staphylococci give positive reactions.
Note: All coagulase negative slides must be confirmed using a tube coagulase test as
the definitive test for S. aureus.

40. Tube Coagulase Test Procedure
* Prepare a 1-in-6 dilution of the plasma in saline (0.85% NaCl) and place 1 ml volumes
of the diluted plasma in small tubes.
* Emulsify several isolated colonies of test organism in 1 ml of diluted rabbit plasma* to
give a milky suspension.
* Incubate the tube at 35°C in ambient air or in a water bath for 4 hours.
* Examine at 1, 2, and 4 hours for clot formation by tilting the tube through 90°.
* Leave negative tubes at room temperature overnight and re-examine.
(This step is essential, for some strains of S. aureus, including many MRSA, produce a
delayed clot which is rapidly lysed at 37°C by the organism’s staphylokinase.)

41. Observation
Read as positive any degree of clot formation. Often the plasma is converted into a stiff
gel that remains in place when the tube is tilted or inverted, but sometimes clots are
seen floating in the fluid.
Coagulase Test
* Coagulase Positive: Clot of any size eg. Staphylococcus aureus
* Coagulase Negative: No clot (plasma remains wholly liquid or shows only a flocculent
or ropy precipitate). eg. Staphylococcus epidermidis
Note: Rabbit plasma is preferable, as it gives better clotting, is free from inhibitors, and is
safe. Human plasma contains sodium citrate as an anticoagulant, and some citrate
utilizing bacteria such as Enterococcus faecalis can destroy the anticoagulant and
cause clotting. False-positive or false-negative results can occur if the plasma is not

42. Reporting
Report as Staphylococcus aureus if the tube coagulase test is positive and the
organism is catalase-positive and a Gram-positive coccus in clusters.
For a negative tube coagulase test from catalase-positive, Gram-positive cocci in
clusters that have creamy white colonies, report as “coagulase-negative
staphylococci.”
A positive slide coagulase test can be reported as S. aureus; however, the test should
be confirmed with a tube test from nonhemolytic or only slightly hemolytic colonies
from sterile sites, such as blood, to separate S. aureus from S. lugdunensis and S.
Schleiferi.
A negative slide coagulase test is not valid and should be followed with a tube test for
confirmation.
Quality Control
Do not use plasma that has not been stored refrigerated or frozen or that appears
turbid.
Perform quality control of coagulase plasma on new lots prior to using them.
Organisms
S. aureus ATCC 25923—coagulase positive

43. Where I can get the plasma?
* Rabbit plasma may be obtained by centrifuging blood to which 0.1% EDTA has been
added as anticoagulant. Alternatively, it may be obtained lypophilized from a commercial
supplier and reconstituted by the addition of water.
* Citrated human plasma may be obtained from a blood bank, but the blood must have
been screened and found free from viral (Hepatitis B, HIV) antigens and antibodies. It
must be handled with the precautions appropriate for all human body fluids.
* Store the plasma in small portions at -20°C and keep a stock of in-used plasma at 4°C
bringing it to room temperature before use.
Limitations
Do not use citrated blood, as false-positive results can occur.
Do not perform coagulase testing from colonies grown on mannitol salt agar.
Methicillin-resistant S. aureus (MRSA) can be deficient in bound coagulase, which
results in a negative slide test.
S. intermedius and S. hyicus may be positive in the tube test; these species are
generally found only in dogs and pigs, respectively.
S. lugdunensis and S. schleiferi give positive slide test but negative tube test. They can
be separated from S. aureus by their strongly positive PYR reaction and from S.
intermedius by a negative tube coagulase test.
Staphylococcus argenteus is a novel staphylococcal species, which is also tube
coagulase-positive and PYR negative. (pyrrolidonyl-β-naphthylamide (PYR))

45. Other coagulase positive staphylococcus
1 Staphylococcus intermedius
2. Staphylococcus hyicus
Coagulase negative staphylococcus
1. Staphylococcus epidermidis ( Normal skin flora)
2. staphylococcus Saprophyticus (Young active female UTI)
How To differentiate
Staphylococcus aureus are Novobiocin – Sensitive, coagulase – Positive
Staphylococcus epidermidis are Novobiocin – Sensitive, coagulase – Negative
staphylococcus Saprophyticus are Novobiocin – Resistant, coagulase – Negative