Exercise no.

1 Bacterial Morphology and Types  Tetrads – cocci that divide in two planes so as to form groups of four
o Gaffkya tetragena
Cocci (spheres) Stain: Gram’s stain

 Staphylococci - cocci that divide into two or more planes to form clusters or
grapelike masses
o Staphylococcus aureus
Stain: Gram’s stain

Bacilli (rods)

 Streptobacilli – bacilli that occur in chains

o Bacillus subtilis
Stain: Gram’s stain

 Streptococci - cocci that divide in a single plane and cling end to end to form chains
o Streptococcus pyogenes
Stain: Gram’s stain

 Diplobacilli - bacilli that occur in pairs

o Snapping - bacilli that bend at the point of division to give two organisms
arranged in the form of a V or inverted V
o Slipping – bacilli that divide and tend to arrange themselves side by side
 Diplococci – cocci that divide in a single plane so as to form pairs  Mycobacterium tuberculosis
o Streptococcus pneumoniae Stain: Acid Fast stain
Stain: Gram’s stain

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  Coccobacilli - short, thick, oval-shaped bacilli  Spirochete - A spiral whose long axis bends when in motion
o Escherichia coli o Borrelia species
Stain: Gram’s stain Stain: Dilute carbol fuchsin
o Treponema pallidum
Stain: Fontana Tribondeau stain

Borrelia sp T. pallidum

 Vibrio – comma-shaped rods

o Vibrio cholera
Stain: Gram’s stain
Exercise no. 2 Bacterial Cell and Special Structures

B. Special structures

 Metachromatic granules
o Granular inclusion bodies; also known as Volutin granules
o Accumulation of metaphosphates formed with the aid of energy yielding
enzymatic reaction
o Usually present in Genus Corynebacterium and Mycobacterium

Corynebacterium diphtheria Mycobacterium tuberculosis

Stain: Loeffler’s Methylene Blue Stain: Acid Fast Stain

Spirals

 Spirillum - a spiral whose long axis remains rigid when in motion

o Campylobacter jejuni
Stain: Gram’s stain

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  Flagella Streptococcus pneumonia Sarcina lutea
o Very fine filamentous appendages; usually seen among rod shaped Stain: Anthony stain Stain: Dilute carbol fuchsin
bacteria
o Originates from the cell membrane
o Composed of flagellin

Proteus vulgaris
Stain: Gray method

Exercise no. 3 Hanging Drop Method

 Hanging drop method – to demonstrate form, arrangement and motility of

microorganisms in the living state
 Vaseline – prevent evaporation of bacterial drop
 Bacterial Spore  Best guide – edge of the drop
o Highly refractile oval of spherical bodies w/in the vegetative cell
Brownian motion - To and fro motion of particles suspended in a liquid; result of physical
o Contains large amount of Calcium dipicolinate – resistance of spores to
forces
heat
o Present in Genus Bacillus and Clostridium
 Gaffkya tetragena
Bacillus subtilis Clostridium tetani
Stain: Gram’s stain Stain: Gram’s stain
True motility - Changing in position in relation to each other. Individual bacteria move across
the slide with varying rapidity

 Proteus vulgaris

Exercise no. 4 Differential Stain

 Gram staining (Hucker’s Method)

 Slime layer and Capsule o Crystal violet – primary stain; 1 minute
o Mucilagenous substance; polysaccharide in nature o Gram’s Iodine – mordant; 1 minute
o Slime Layer – only small amount is present o Acetone alcohol - decolorizer; drop by drop
o Capsule - forms a definite layer o Safranin – counterstain; 30 seconds

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Staining reaction: Gram (+) Staining reaction: Acid fast Staining reaction: Non acid fast

Staphylococcus epidermidis Bacillus subtilis Mycobacterium tuberculosis Bacillus subtilis

Morphology: staphylococcus Morphology: streptobacilli Morphology: diplobacilli Morphology: streptobacilli

Exercise No. 5 Method of Obtaining Pure Culture

A. Streak Plate Method

Staining reaction: Gram (-)  4 quadrant streaking
 Nutrient agar
Branhamella catarrhalis Escherichia coli  24 hour incubation at 37 degrees upside down; prevent separate colonies to join
Morphology: diplococci Morphoplogy: coccobacilli due to moisture

Specimen: (Obtained using a sterile cotton swab and sterile NSS)

 Acid Fast Staining (Ziehl-Neelsen Method) 1. Skin (arm) 6. Area in between toes
o Carbol fuchsin - primary stain; 3-5 minutes (heating), 5 minutes without 2. Teeth 7. Throat
heating 3. Ear 8. Face
o Acid alcohol - decolorizer 4. Armpit 9. Hair
o Methylene blue - counterstain; 30-45 seconds 5. Nostril 10. Palm

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 B. Pour Plate Method o All tubes are added with impregnated discs and incubated at 37 °C for 24
 Gaffkya tetragena with a predetermined dilution hours
 1 ml of suspension is pipetted in the petri dish with the agar. Bacteria Interpretation
 Allow agar to harden after swirling and incubate for 24 hours at 37 degrees upside Boiling 62°C Autoclave Hot Air
down B. subtilis Not killed Not killed Killed Killed
E. coli Killed Killed Killed Killed
Gram staining:
Bacillus subtilis Escherichia coli

Exercise No. 6 Effects of Physical Agents on Microorganisms

Exercise No. 7 Action of Disinfectants on Microorganisms
 Bacillus subtilis and Escherichia coli suspensions  5 Lactose broths with Durham’s fermentation tube containing Andrade’s indicator
 Filter paper discs impregnated with B. subtilis and E. coli Pink in the presence of acid
 Nutrient broths
 Gram stain bacterial suspension Disinfectant:
 CONTROL  3% H2O2
 BOILING  70% Ethanol
 62 °C – 30 minutes then placed in ice cold water  95% Isopropyl alcohol
 AUTOCLAVE  Zephiran Chloride 1:1000
 HOT AIR  Sodium Hypochlorite 1%
 5% Phenol
 5% Lysol
 10% Betadine
 Merthiolate
 1:1000 Mercuric Chloride
Results: The organisms where killed in 5 minutes

**E.coli; only control is turbid Durham’s tube

(gas is present)

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Exercise 8. Antimicrobial Susceptibility test: Diffusion Test Procedure

 Mueller Hinton Infusion Agar

 Antibiotic discs
 Sterile cotton swab
 Sterile NSS
 Forceps, inoculationg wire loop
 Standard: Barium Sulfate Comparison Standar (0.5 mL of 0.048M BaCl2 to 99.5 mL
of 0.36 N H2SO4) McFarland Barium Sulfate no. 0.5
6
o Approximate cell density – 1.5 x 10 CFU/mL
 Streaking method - Very Close Streaking at 60° turning 3 times; 1 final sweep to
agar rim
 Allow plate to stand for 3-5 minutes (not more than 15 minutes)
 Antibiotic discs should be 15 mm from the edge of the agar and 20 mm apart from
each other to prevent overlapping of zone of growth inhibition
Exercise no. 9 Staphylococcus
 Within 15 minutes after the discs are applied, invert and incubate at 37°C for 16-18
hours
 Staphylococcus aureus and Staphylococcus epidermidis
 Examine and measure the diameter to the nearest mm
 Blood Agar plates, Trypticase Soy Agar, Mannitol Agar
 Plasma (medium for coagulase test)
 H2O2; Sterile NSS
Results: ST
Ampicillin Chloramphe- Gentamicin Penicillin Trimenthoprim-
1 Meeting
nicol sulfamethoxazole  Make a smear and do gram stain of the organisms
 BA plate = 4 quadrant streaking; TSA slant = Simple Streaking; incubate at 37°C for
mm Int. mm Int. mm Int. mm Int. mm Int. 24 hours
Escherichia coli 17 S 31 S 22 S 11 R 30 S  Describe colonial characteristics and haemolytic property
Staphylococcus 22 S 27 S 23 S 23 I 30 S
aureus
Salmonella typhi 26 S 29 S 25 S 23 I 35 S
Shigella 7 R 11 R 23 S 7 R 28 S
dysenteriae
Pseudomonas 6 R 12 R 21 S 6 R 6 R
aeroginosa
Proteus vulgaris 24 S 22 S 22 S 26 I 27 S
Klebsiella 6 R 27 S 20 S 6 R 6 R
pneumoniae
Salmonella para A 26 S 34 S 29 S 13 R 33 S
Enterobacter 6 R 10 R 24 S 6 R 6 R
cloacae
Salmonella 27 S 31 S 21 S 24 I 26 S
enteritidis
**Results based on MT3C

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Slide Coagulase
 Divide glass slide into C (control) and T (test) Catalase Production
 Place a small drop of NSS on both side  Add 1 mL of H2O2 into TSA slant
 Make a heavy suspension with the isolated colonies on BA; Add loopful of Plasma  Observe for immediate appearance of gas bubbles
to T; observe clumping within 10 seconds
 Cell bound coagulase

Tube Coagulase
 Small test tube w/ 0.5 mL fresh human plasma; Inoculate the plasma with a loopful
of bacteria from BA Bacteria Gm. Stain rxn & Mx Colonial morph. Pigment
 Incubate at 37°C and observe for clotting at intervals of 30 minutes for 4 hours. morph.
 Free coagulase Staphylococcus aureus Gram (+) Small, round, glistening, Golden
staphylococci small yellow
Staphylococcus Gram (+ White, circular, smooth, white
epidermidis staphylococci small

Hemolysis on BA Slide coagulase Tube coagulase Catalase test Mannitol fermentation

Β – hemolysis + + + Acid
γ - hemolysis - - + NC

Mannitol Fermentation
 Get an inoculums from BA using a wire needle and stab the agar until few mm from
the bottom; incubate at 37°C for 24 hours
 Observe color change (pink)

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Exercise 10. Alpha-Hemolytic Streptococci Optochin Sensitivity test
 Inoculate BA plate heavily by streaking closely with 2 or 3 colonies as inoculums
 Streptococcus pneumonia and Streptococcus viridians  Place optochin disc at center of inoculated pplate
 BA plates; Inulin Agar; Skimmed milk; 5 µg Optochin disc (ethylhydrocupreine HCl);  Incubate plate aerobically at 37°C
10% solution of sodium deoxycholate; 1% aqueous crystal violet; 20% copper  Read results after 24 hour incubation
sulphate; Sterile NSS  Observe for zones of growth inhibition
 Do gram stain of the organisms o Zones equal or greater than 14 mm surrounding a 6 mm disc - positive
 Inoculate BA and skimmed milk with the organism; incubate both in 37°C inside a o Zones equal or greater than 16 mm surrounding a 10 mm disc – positive
candle jar for 24 hours; describe colonial appearance and haemolytic property  Presumptive ID for S. pneumoniae
Capsule Staining (Anthony Method)
 Make a thin smear; air dry
 Stain with 1% aqueous crystal violet for 2 minutes
 Wash with a solution of 20% copper sulphate
 Air dry in vertical position

Bile Solubility Test

 Add colonies to a tube with 2 ml NSS until turbid
 Divide suspension into 2 tubes
 One tube is Control; to the other test tube add a few drops of 10% solution of
Sodium deoxycholate
 Observe for clearing or lysis which occurs in 5-10 minutes. Compare turbidity with
Colonial morph. Hemolysis Gm. Stain rxn & Capsule
the control tube
Mx morph
Streptococcus Pinpoint, round w/ greenish zone α- Gram (+) Not
viridans of hemolysis,transparent Hemolysis streptococci capsulated

Inulin ferment Bile Solubility Optochin test

Acid Bile insoluble Resistant
**results based on MT3C

Exercise 11. Beta- Hemolytic Streptococci

Inulin Fermentation
 Inoculate Inulin agar w/ a wire needle by stabbing until few mm from the bottom
 Incubate at 37°C and read after 24-48 hours. Observe for color change in the
medium
 Stock cultures of group A beta-hemolytic streptococci and nongroup A beta-
hemolytic streptococci
 BA plates; 0.04U Bacitran disc
 Make a smear from stock culture, gram stain
 Inoculate BA plate using wire loop; Multiple interrupted streaking, 2-3 stabbings at
the end of last line; Incubate at 37°C and take note of colonial and haemolytic
properties of the organism

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Bacitracin test Pathogenic Neisseriae
 From the same stock culture, obtain heavy inoculums (3-4 colonies) and inoculate
½ of theplate by streaking as follows:  Neisseria gonorrheae
o Make a vertical line across ½ of the BA plate. Streak closely across this
vertical line with the loop in a vertical position
 Place Bacitracin disc on center of inoculated area
 Incubate BA plate aerobically at 37°C; read result after incubation; examine for a
Zone of growth inhibition around bacitracin disc; any zone of growth inhibition is
indicative of bacitracin susceptibility.

Colonial morph. Hemolysis Gm. Stain rxn & Mx Bacitracin

morph susceptibility
Streptococcus Pinpoint, round, β - hemolytic Gram (+) Sensitive
pyogenes transluscent streptococci
w/clear zone of
hemolysis
Group A β Pinpoint, round, β - hemolytic Gram (+) Sensitive
streptococci transluscent streptococci

 Nesseria meningitidis
Exercise no. 12 Pathogenic and Nonpathogenic Neisseria

 Neisseria meningitides and Neisseria gonorrheae (pathogenic)

 Neisseria sicca and Branhamella catarrhalis(non-pathogenic)
 Chocolate Agar, Nutrient agar; Glucose, Maltose, sucrose, lactose agars
 Oxidase rgt. ( 1% p-aminodimethylaniline oxalate

G M S L

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  Branhamella catarrhalis
Nonpathogenic Neisseriae

 Make gram stain

 Inoculate CA and NA by 4-quadrant streaking; Glucose, sucrose, maltose and
lactose are stabbed until few mm from the bottom with the wire needle
 Incubate all at 37°C inside candle jar except NA which is incubated aerobically;
examine plates and tubes after 24 hours
 Make a smear on the isolated colony on the NA plate

Oxidase test
 Add a few drops of 1% solution of p-aminodimethylaniline oxalate
o Turns pink to red to purple then black in 10-30 seconds

 Neisseria sicca

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