MICROBIOLOGY AND PARASITOLOGY

LABORATORY NOTES/ ACTIVITIES

PART I: BASICS OF MICROORGANISM IDENTIFICATION

A. MORPHOLOGY
SHAPE  Coccus/cocci- round/ circular/spherical
 Bacillus - rod shaped
 Pleomorphic- vary in shape
o Enlarged rod (Fusobacterium)
o Comma form
o Club rod (Corynebacteriaceae)
o Helical form (Helicobacter pylori)
 Spiral
o Vibrio - straight rod or with single rigid curve
o Spirillum - rigid helical rod
o Spirochete - flexus helical rod
o Filamentous
o Corkscrew form (Borrelia burgdorferi)
ARRANGEMENT  Pairs: Diplococci, diplobacilli
 Chains: Streptococci, staphylococci
 Grape-like-clusters: staphylococci
 Group of four: tetrads (e.g. Peptococcus)
 Packets of eight: cuboidal (e.g.Sarcina)
 Palisades: (e.g.Corynebacterium)
SIZE  Unit of measurement- micrometer (1 micrometer or um (formerly micron or
u)
 Haemophilus - smallest pathogenic bacillus (0.2 x 0.5 um)
 Bacillus anthracis - largest pathogenic bacillus (1 x 3-10 um)
CAPSULE  Outermost layer, if present
 Encapsulated Bacteria: “Some Killers Have Pretty Nice Capsule”
o Streptococcus pneumoniae
o Klebsiella pneumoniae
o Haemophilus influenzae
o Pseudomonas aeruginosa
o Neisseria meningitidis
o Cryptococcus neoformans (fungi)
 Virulence factor (VF): K antigen → prevents phagocytosis
 Mucoid and slimy polysaccharide layer
 Special staining methods
o Hiss stain
o India ink stain (negative staining)
CELL WALL  Cell wall (Also known as peptidoglycan)
 Functions:
o Protects bacteria against osmotic pressure
o Gives shape to the bacterial
o Confer the grams reaction of the bacteria
o A usual target of antimicrobial drugs (e.g. penicillins, cephalosporins)
 Important for identification
I. Gram-positive cell wall
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II. Gram -negative cell wall
III. Acid-fast cell wall

GRAM POSITIVE V.S GRAM NEGATIVE

GRAM POSITIVE GRAM NEGATIVE
Peptidoglycan Thick Thin
Lipoteichoic acid Present Absent
LPS contents None High
Periplasm Absent Present
Cytoplasmic Present Present

B. MICROSCOPY

ORGANISM BFM FLUORESCENCE DARK FIELD ELECTRON

GROUP MICROSCOPE MICROSCOPE MICROSCOPE
BACTERIA + + + -
FUNGI + + - -
PARASITE + + - +
VIRUSES - + - +

 BRIGHT FIELD MICROSCOPE

o Gram staining, Acid fast stain, KOH
o Most common microscope
 DARK FIELD MICROSCOPE
 PHASE-CONTRAST MICROSCOPE
o Good for KOH mount
 FLUORESCENT MICROSCOPE
o Requires fluorescent stain
 ELECTRON MICROSCOPE
o Transmission Electron Microscope
o Scanning Electron Microscope
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C. STAINING PROCEDURES
o GRAM STAINING TECHNIQUE

Gram Staining is the common, important, and most used differential staining techniques in microbiology.
This test differentiate the bacteria into Gram Positive and Gram Negative Bacteria, which helps in the
classification and differentiations of microorganisms.

PRINCIPLE:

Bacteria with thick cell walls containing teichoic acid and retain the crystal violet- iodine complex dye after
decolorization. Other bacteria with thinner cell walls containing lipopolysaccharide do not retain the dye
complex.

REAGENTS GRAM POSITIVE GRAM DURATION

NEGATIVE
Primary stain Crystal violet Purple color Purple color 1 minute
Mordant Gram’s Iodine Purple color Purple color 1 minute
Decolorizer Acetone and Purple color Colorless Quick and rinse
Alcohol (95%)
Secondary stain Safranin Purple color Red/Pink color 30 seconds

Note:

- This is the most important diagnostic tool in treating patients with clinical infections in the emergency
department.

- Once stained, examined using the 10x objective lens (LPO). then the smear should be examined using
100x objective lens (OIO) for phagocytes, bacteria, and other cellular material.

o ACID- FAST STAIN

Principle: Primary stain binds to mycolic acid in the cell walls of mycobacteria and is retained after
the decolorizing step with acid-alcohol

Procedure:

1. Crush between slides a drop of the purelent or mucoid material: or a loopful of the specimen or
culture is smeared in to a clean slide. Air dry the smear then heat fixes on the burner flame for a
few seconds.
2. Place the slide on a staining rack and flood with the carbol fuschin solution: apply a low steaming
heat and maintain for 4-5 minutes.
3. Cool the slide for a few seconds then wash with water,
4. Decolorizer the smear with acid alcohol until no further stain can be rinse off.
5. Rinse briefly in tap water, and then counter stain with methylene blue solution for 20-45 seconds.
6. Rinse, drain, dry by clothing.
7. Examine the stained smear under the microscope using the LPO and the OIO.
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REAGENTS ACID FAST NON-ACID FAST
ZIEHL-NEELSEN KINYOUN ORGANISM ORGANISM
( hot method) (cold method)
PRIMARY STAIN Carbol fuschin Pink/Red Pink/Red
MORDANT Heat/Steam Tergitol/Phenol Pink/Red Pink/Red
DECOLORIZER Acid alcohol (3% HCl in 95% Ethanol) Pink/Red Colorless
SECONDARY Methylene Blue Malachite Pink/Red Blue/Green
STAIN green/Methylene
Blue

AFB GRADING (NATIONAL STANDARD)

Negative No AFB/300 fields
Doubtful 1-9 AFB/100 fields
1+ 10-99 AFB/100 fields
2+ 1-10 AFB/field in at least 50
fields
3+ >10 AFB/field in at least 20
fields

OTHER STAINING TECHNIQUES

Bacteria with MYCOLIC  Mycobacterium, Rhodococcus, Nocardia Corynebacterium,
ACID Tsukamurella, Gordonia and Dietza
Auramine- Rhodamine  Auramine O with or without Rhodamine B
( TRUANT'S)  0.5 % acid-alcohol
 Mycobacteria appear as slender rods that flouresce bright yellow
(Auramine O) or yellow red (Auramine- Rhodamine B) againts a dark
background.
Pappenheim's stain  M. tuberculosis- RED
 M. semegmatis- BLUE
Baungarten's stain  M. tuberculosis BLUE
 M. leprae- RED
Schauffer- Fulton  Spore formers
Feulgen  Nuclei/ DNA
Spengler's stain  For color blind individuals AFS + organisms will appear BLACK
Modified Acid Fast  Decolorizer: 1% Sulfuric acid
 Secondary stain: 0.5% Potassium permanganate
 AFS+: Yellow *MRNGTG + L. micdadei
 Parasite: Isospora, Cyclospora, and Cryptosporidium
Spore stain  Composition: Calcium dipicolinate
 Genera: Bacillus and Clostridium
Flagellar stain  MORDANTS such as Tannic acid or potassium alum prior to staining
 Stains: Fisher-Conn, Leifson's, Gray's
Capsule stain  Hiss, Gin's, Welch's, Muir's, India ink
Silver stain  Warthin-Starry and Steiner ( silver impregnation stain)- demonstrates
spirochetes in formalin-fixed tissue sections
 Modified Dieterle- demonstrates Borrelia burgdorferi
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D. SMEAR PREPARATION

The process of making a smear preparation is an important skill in the microbiology laboratory and is
usually the first step in most staining procedures. The quality of the smear will directly affect the quality of
the subsequent staining procedure. The smear preparation differs slightly depending on the specimen or
culture.

Supplies:
 Personal protective equipment
 Sharps container
 Biological waste container
 Microscope slides with frosted-edge
 Pencil or wax pencil
 Sterile saline or water
 Sterile pipettes
 Loops or applicator sticks
 Slide warmer, Bunsen burner, or methanol

Instructions
1. Be sure to wear appropriate personal protective equipment (PPE) to include gloves, laboratory coat, and
face and eye protection, as indicated in your laboratory's SOP and safety manual.
2. Obtain a clean microscope slide with a frosted edge.
3. Label the frosted edge appropriately with the sample identification.
4. Transfer specimen or culture to the center of the slide.
a. Clinical Specimen: Prepare a thin layer of cells on the slide. Refer to your laboratory’s procedure
according to different specimen types.
b. Broth Culture: Using a sterile pipette, transfer 1-2 drops to the slide. Spread the drop into a thin,
even smear.
c. Culture from solid media: Using a sterile pipette, add one drop of sterile saline or sterile water to
the center of the microscope slide. Aseptically pick a small amount of an isolated colony with a loop and
gently mix into the drop of sterile saline or water using circular motions. Mix evenly to make a thin smear.
5. Allow the smear to air dry completely.
6. Fix the smear to the slide using heat fixation or methanol fixation according to your laboratory’s
procedure.
7. Allow the slide to cool to room temperature or air dry.

Note: Do not drag the 40X objective through the oil.

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E. CULTURE MEDIA
 Nutrient material prepared for microbial growth
o Agar - complex polysaccharide derived from marine algae
o Colony - visible growth of microbes on the surface of a medium
Simple For non-fastidious organisms
Complex Made up of nutrients from diff
sources
Enriched To increase very small numbers to o Milk agar
detectable levels; for fastidious o Blood agar
Anaerobic/ For obligate anaerobes o Thioglycolate agar
Reducing
Selective Suppress growth of unwanted & o Bismuth sulphite agar
encourage the growth of desired o Saboraud’s dextrose agar
ones o Brilliant green agar (Salmonella)
o Middlebrook (Mycobacterium)
o Lowenstein-Jennsen (Mycobacterium)
o Colistin, Nalidixic acid (g +)
Differential Distinguish colonies on same plate o Blood agar (Streptococci α, β, γ)
Combined Selective o Mannitol salt agar (S.aureus)
and Differential o MacConkey agar

CULTURE MEDIA
 Saboraud’s dextrose agar/PDA - fungi
 Lowenstein-Jennsen - M. tuberculosis
 Loeffler’s - diptheriae
 Thiosulfate-Citrate-Bile Salts-Sucrose – V. cholerae
 Skirrow’s - H. pylori, C. jejuni
 Chocolate/Blood - H. influenza
 L-cysteine - Legionella pneumophila
 Thayer-Martin VCN - Neisseria
 Fletcher’s - Leptospira interrogans
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NAME: DATE:

COURSE/YEAR/SECTION: SCORE:

PART I: BASICS OF MICROORGANISM IDENTIFICATION

(THE GRAM STAINING TECHNIQUE)
OBJECTIVE:
Became adept at Gram staining bacterial smears with consistent gram reactions and gain a general
understanding of some theoretical explanations of differing gram reactions.

MATERIALS:

Gram staining solution:

Solution of Crystal Violet
Gram’s Iodine Solution
95% Ethyl Alcohol
Safranin Solution
Staining equipment (Staining rack, wash bottle, surgical gauze, droppers, forceps)
Surgical gloves
Unstained smears
Running water source

ACTIVITIES:

1. Unstained smear
2. Heat fixes the smear and places it on staining rack
3. Apply the following reagents/solutions in order:
a. Flood the slide with CRYSTAL VIOLET for about 1 minute.
b. Drain, remove the stain and wash carefully with water. Do not allow force of water to touch the
smear.
c. Then flood the smear with GRAM’S IODINE SOLUTION and allow to set forth for 1 minute.
d. Decolorize with 95% ALCOHOL until most of the stain comes off from the smear. This requires at
most 30-45 seconds.
e. Rinse the slide with tap or running water, then counter stain with SAFRANIN SOLUTION for 45
seconds.
f. Finally, rinse with tap or running water, air dry or blot dry between surgical gauze.
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DRAW AND LABEL:

Draw the procedures in order of the Gram staining technique.

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LABORATORY NOTES/ ACTIVITIES
QUESTIONS FOR RESEARCH:

1. Discuss the various theories on the Gram Stain

2. List factors that could cause variability in gram reactions of an organism.

3. Describe the cell wall of a gram-positive bacteria and what is its purpose.

4. What is the purpose of a secondary stain in gram staining?

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5. Describe the cell wall of acid fast bacilli.

6. Give specific examples of the following (3-5 each).

A. Gram positive cocci

B. Gram negative cocci

C. Gram positive bacilli

D. Gram negative bacilli

 MICROBIOLOGY AND PARASITOLOGY
LABORATORY NOTES/ ACTIVITIES
NAME: DATE:

COURSE/YEAR/SECTION: SCORE:

PART I: BASICS OF MICROORGANISM IDENTIFICATION

(THE ACID-FAST STAINING METHOD)
OBJECTIVE:
Know the principle and technique in acid-fast staining method using Ziehl-Neelsen procedures, and
able to differentiate between acid-fast organisms.
MATERIALS:
Acid-Fast staining solutions
Carbol fuschin solution
Acid-alcohol (Decolorizer)
Methylene blue solution
Glass slides, inoculating loop, staining rack, filter paper
Microscope, bunsen burner, water trough
Specimen: Sputum (or any available culture plate from the clinical lab.)

ACTIVITY:
1. Crush between slides a drop of the purelent or mucoid material: or a loopful of the specimen or
culture is smeared in to a clean slide. Air dry the smear then heat fixes on the burner flame for a
few seconds.
2. Place the slide on a staining rack and flood with the carbol fuschin solution: apply a low steaming
heat and maintain for 4-5 minutes.
3. Cool the slide for a few seconds then wash with water,
4. Decolorizer the smear with acid alcohol until no further stain can be rinse off.
5. Rinse briefly in tap water, and then counter stain with methylene blue solution for 20-45 seconds.
6. Rinse, drain, dry by clothing.
7. Examine the stained smear under the microscope using the LPO and the OIO.
8. Interpretation:

Ziehl-Neelsen Acid Fast Non Acid Fast Organism

(Hot method) Organism
Primary Stain Carbol fuschin PINK PINK
Mordant Heat PINK PINK
Decolorizer Acid alcohol PINK COLORLESS
(5% Sulfuric acid)
Secondary Methylene Blue PINK BLUE
Stain

ACID-FAST ORGANISMS: stain red or pink

NON-ACID FAST ORGANISMS: stain blue

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QUESTIONS FOR RESEARCH:

1. Differentiate between the “hot method” and “cold method” of acid-fast staining.

2. What is the Fluorochrome staining technique for Mycobacterium?

3. What chemical constituent is responsible for acid fast of Mycobacteria species?

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4. Aside from Mycobacterium, name other groups which are considered acid-fast or partially acid-fast.

5. Give the ingredients and preparation of the acid-fast staining solution.