LESSON 2: BACTERIAL IDENTIFICATION AND PROCESSING
I. Learning Objectives
At the end of the lesson, students should be able to:
A. Explain the parts of a microscope.
B. Differentiate types of microscopes.
C. Define terms related to microscopy.
D. Discuss and identify the different types of specimen that is received at Bacteriology
section for processing.
E. Discuss the different types of bacterial staining and their significance.
F. Explain the principles of Gram staining and Acid-fast staining.
G. Identify the various measures to achieve proper staining of bacteria.
H. Discuss the types of culture media and their purpose.
I. Identify various inoculation techniques for the proper isolation of microorganisms.
J. Differentiate the stages of bacterial growth, including the generation time.
II. Concepts and Information from the PowerPoint
Microscopy
Microscope is essential in magnifying microorganisms to be visualized with naked eye.
Ideally, it should be PARFOCAL.
Types of Microscope
A. Light Microscope
▸ Uses glass lenses to bend and focus light rays, thus creating magnified images
of objects.
▸ The visible light passes through the specimen and through a series of lenses that
reflect light, resulting to magnification of the organisms.
A1. Bright Field Microscope
▸ Most commonly used light microscope in a clinical laboratory.
▸ Forms a dark image against a brighter background.
▸ Magnification of the Lens:
▸ Ocular (Eyepiece) Lens- 10x
▸ Objective Lenses
▸ Low Power Objective (LPO)- 10x
▸ High Power Objective (HPO)- 40x
▸ Oil Immersion Objective (OIO)- 100x
▸ Purposes of Cedar Wood Oil:
▸ Enhances the resolution and used to fill the space between
the objective lens and glass slide.
▸ Prevents light rays from dispersing and changing their
wavelength after passing through the samples.
▸ Parts of Bright Field Microscope
▸ Ocular (eyepiece) lens
▸ Observes the specimen and re-magnifies the image formed by the
objective lens.
▸ Lens must be small to produce a high magnification with good
resolution.
▸ Arm
▸ Used for holding the microscope.
▸ Revolving Nosepiece
▸ Holds the objectives.
▸ Rotating this part easily changes the magnification of the
objectives.
▸ Objective Lens
▸ Primary lens that magnifies the specimen.
▸ Most important part of the microscope that produces a clear image
of the specimen.
▸ The higher the magnification number of an objective, the greater is
the feature or detail seen in a specimen.
▸ Coarse Focus Adjustment Knob
▸ It is rotated to obtain a coarse focus by moving the stage up and
down.
▸ Fine Focus Adjustment Knob
▸ It is rotated to obtain a fine focus by moving the stage up and down.
▸ Mechanical Stage
▸ Where the specimen is placed.
▸ Specimen Holder
▸ Holds the glass slide in position.
▸ Condenser
▸ Focuses the light from the illumination source through the specimen
and magnified by the objective lens.
▸ Incorporates a lens which collects illumination light on the stage so
that the objective lenses can perform at full capability.
▸ Iris Diaphragm Dial
▸ It controls the amount of light entering the condenser.
▸ Base
▸ Supports the entire microscope.
A2. Phase Contrast Microscope
▸ Permits detailed examination of internal structures in living organisms.
▸ Identifies medically significant fungi frown in culture.
▸ The phasic differences are seen as different degrees of brightness.
▸ Used to examine unstained living cells.
A3. Fluorescent Microscope
▸ Involves excitation of fluorochromes using light.
▸ Uses fluorochromes (dyes).
▸ Utilized in the observation of chlamydiae, legionellae, mycobacteria, and fungi.
▸ Examples of DYES: acridine orange, auramine and rhodamine, calcofluor white,
and fluorescine isothiocyanate (FITC).
A4. Dark Field Microscope
▸ Uses dark field condenser that blocks away light that can directly enter the
objective.
▸ Directs the light to hit the specimen at an oblique angle.
▸ The background becomes a dark field in which organisms appear extremely
bright.
▸ Detect spirochetes (T. pallidum)
B. Electron Microscope
▸ Uses electrons to visualize small objects.
▸ Useful for studying the morphology of bacteria.
▸ Has a built-in camera to capture images of the cells in black and white
transmission electron micrographs.
▸ Advantage: Objects smaller than 0.2µm can be visualized with 100,000x
magnification.
B1. Transmission Electron Microscope
▸ Allows visualization of the internal structures of cells.
▸ Has the greatest resolution that is approximately 1,000 times higher than the light
microscope
▸ Used to examine very thin specimens and organisms since it can magnify a
specimen a million times.
B2. Scanning Electron Microscope
▸ Scans the surface of the cells or specimens.
▸ 3-dimensional images
▸ Focuses a beam of high-energy electrons through specimen
▸ Magnification reaches up to approximately 30,000x
Specimen Types
A. Blood
▸ Highest concentration of microbes: during the height of fever
▸ Disinfect venipuncture site with 70% alcohol then betadine
▸ 10-20mL per set is collected in adults
▸ 1-5 mL in children
▸ 0.5-1mL in neonates
▸ Bacteremia: Bacteria in blood
▸ Septicemia: Increase in numbers of bacteria in the blood causing harm to the
patient
B. Cerebrospinal Fluid (CSF)
▸ Rapid testing is recommended
▸ Cytocentrifuge to collect sediment for staining or culture
▸ The most common significant isolates found in CSF are Neisseria meningitidis,
Streptococcus agalactiae, Staphylococcus aureus and Listeria monocytogenes
C. Throat and Nasopharyngeal Specimen
▸ Most abundant normal flora: Streptococcus viridans
▸ Most common pathogen: Streptococcus pyogenes
D. Stool
▸ If bacterial infection is suspected 3 specimens must be collected (once a day for
3 days)
▸ Most common transport medium is Cary-Blair
E. Sputum
▸ Sterile, screw-cap container
▸ Rinse mouth or gargle with water, instruct to cough deeply into container
▸ Used to diagnose lower respiratory tract infections
▸ A direct gram stain is performed to determine the quality of specimen
▸ A general rule for acceptable specimen is <10 squamous epithelial cells and >25
PMNs/low power field
▸ Common significant sputum isolates: Streptococcus pneumoniae, Klebsiella
pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila
F. Urine
▸ Sterile, screw-cap container
▸ Clean area with soap then rinse with water. Discard first few mL of urine, start
collecting midstream
▸ Escherichia coli- most common agent of UTIs in both ambulatory and
hospitalized patients
▸ Staphylococcus saprophyticus- 2nd most common; most common cause of UTI in
young, sexually-active females
▹ Midstream clean-catch- specimen of choice for urine culture
▹ Catheterized urine
▹ Suprapubic urine
G. Genital
▸ For diagnosis of venereal diseases or STDs
▸ Infections caused by Treponema pallidum, Neisseria gonorrhoeae, Chlamydia
trachomatis and Herpes Simplex Virus
▸ Specimens: Cervical (female), urethral (male), rectal and throat swabs
▸ The vagina contains normal flora that changes with age: Lactobacillus spp.
(during childbearing years), Staphylococci and Corynebacterium (early and late
in life)
H. Lesion, Wound Abscess
▸ Superficial: swab along the outer edge
▸ Deep: aspirate with needle and syringe
*Note: When multiple specimens arrive at the same time, priority should be given to
those that are most critical such as CSF, tissue and blood
Bacterial Staining
STAINS are chromogenic solutions that are essential in the microscopic identification
and differentiation of bacterial cells.
A. Objectives of Staining
▸ To determine the morphology of bacteria
▸ To differentiate groups of bacteria
▸ To identify organisms with special structures
B. Staining Techniques
▸ Simple Staining
▸ A single stain is used.
▸ Directed towards coloring the forms and shapes of the cells.
▸ Example: Use of methylene blue.
▸ Differential Staining
▸ Divides bacteria into separate groups.
▸ Directed towards coloring the components of the elements present.
▸ Examples: Gram staining; Acid-fast staining (AFB)
▸ Negative Staining
▸ Utilized to demonstrate the presence of diffuse capsule surrounding some
bacteria.
▸ Excellent technique for studying bacterial gas vacuoles and viral
morphology.
▸ Results in the bacteria appearing as light colored bodies against a dark
background since the cell surface repels the acidic stain as a result of
bacterial cells being negatively charged.
▸ Example: Use of India ink or Nigrosin dye.
D. Gram Stain
▸ Most commonly used differential stain in the clinical microbiology laboratory.
▸ Utilizes crystal violet as the primary stain while safranin is the secondary stain
or counterstain.
▸ Iodine acts as the mordant while acetone-alcohol mixture acts as the decolorizing
agent.
▸ Principle of Gram Staining:
▸ Bacteria with thick cell walls containing teichoic acid and thick
peptidoglycan layer retain the crystal violet iodine complex dye after
decolorization and appear purple, which means that they are Gram
positive.
▸ Other bacteria with thinner cell walls that contain lipolysaccharides do not
retain the dye complex and appear deep pink or red, which mean that they
are Gram negative.
▸ Gram Staining Procedure
▸ 1. Flood slide with crystal violet for 1 minute.
▸ 2. Wash with water.
▸ 3. Apply mordant (Gram’s iodine) for 1 minute.
▸ 4. Wash off with water.
▸ 5. Add 95% alcohol or acetone until it runs clear.
▸ 6. Wash off with water.
▸ 7. Counter-stain with safranin for 45 seconds.
▸ 8. Wash off with water.
▸ General Rules of Gram Staining
▸ All cocci are Gram positive except Neisseria, Veilonella, Branhamella
and Moraxella.
▸ All bacilli are Gram negative except for Bacillus, Lactobacillus, Listeria,
Actinomyces, Clostridium, Corynebacterium, Mycobacterium,
Erysipelothrix and Nocardia.
E. Acid-fast Stain
▸ Used to stain bacteria that have high lipid contents in their cell walls (mycolic
acid).
▸ Common isolate detected M. tuberculosis
▸ Also called AFB or acid fast bacilli staining
▸ Utilizes carbol fuchsin as the primary stain and methylene blue or malchite
green as the secondary stain.
▸ The cell wall of acid-fast bacteria resists the acid-alcohol (hydrochloric acid-
ethanol mixture) decolorization step.
▸ Heat is applied as a mordant in the Ziehl-Neelsen method while tergitol is used
in Kinyoun method.
▸ Principles of Acid-fast Staining
▸ The primary stain binds to the mycolic acid in the cell walls of the acid-fast
bacteria, like in mycobacteria, and is retained after decolorizing with acid
alcohol.
▸ Acid-fast bacilli (AFB) retain the primary stain and are deep pink or red
while non-AFB are either blue or green (methylene blue or malachite
green counterstains).
ZIEHL-
KINYOUN/ NON ACID-
NEELSEN/ ACID-FAST
COLD FAST
HOT ORGANISM
STAINING ORGANISM
STAINING
PRIMARY STAIN Carbolfuchsin Carbolfuchsin Stains red Stains red
Remains Remains
MORDANT Steam Tergitol/Phenol
red red
Remains Becomes
DECOLORIZER Acid Alcohol Acid Alcohol
red colorless
Methylene
Methylene
blue/ Remains Stains blue/
COUNTERSTAIN blue/ Malachite
Malachite red green
green
green
▸ Acid-fast Staining Procedure
▸ 1. Carbol fuchsin for 5 minutes.
▸ 2. Wash with water.
▸ 3. Decolorize with acid alcohol for 30 seconds.
▸ 4. Wash off with water.
▸ 5. Counter-stain with methylene blue for 3 minutes.
▸ 6. Wash off with water.
▸ Notes to remember in Acid-fast Staining
▸ Mycolic acid renders the cells resistant to decolorization, thus the term
“acid-fast”.
▸ Acid alcohol decolorizing agent is composed of hydrochloric acid and
ethanol.
Culture and Culture Media
CULTURES are the growth of microorganisms in a culture medium. Utilizing effective
and appropriate culture medium for growth, transport, and storage facilitates the study
of microorganisms.
A. Classification of Culture Media
▸ According to Consistency
▸ 1. Liquid Medium
▸ Does not contain any amount of agar.
▸ Allows the growth of aerobes, anaerobes, and facultative
anaerobes.
▸ Examples: brain heart infusion (BHI); trypticase soy broth (TSB);
and thioglycollate.
▸ 2. Semi-solid Medium
▸ Contains 0.5% to 1% agar.
▸ Used to observe bacterial mobility and detect indole and sulfide
production.
▸ Example: sulfide indole motility (SIM) medium.
▸ 3. Solid Medium
▸ Contains 2% to 3% agar.
▸ Examples: triple sugar iron (TSI) agar; MacConkey (MAC) agar,
blood agar plate (BAP), and chocolate agar plate (CAP).
▸ According to Use
▸ 1. Simple Media, General Purpose Media, and Supportive Media
▸ Routinely used in the laboratory without additional supplements.
▸ Supports the growth of most non-fastidious bacteria.
▸ Fastidious-complex nutritional req’t (blood, thiosulfate, cysteine)
▸ Non-fastidious- basic nutritional req’t (carbon, nitrogen, water)
▸ Usually composed of meat and soybean extracts.
▸ Examples: nutrient agar (NA), nutrient broth (NB), and TSB.
▸ 2. Enrichment Media (Liquid-type Media)
▸ Used to propagate the growth of a certain group of bacteria from a
mixture of organisms.
▸ Contain specific nutrients without additional supplements.
▸ Examples: alkaline peptone water, selenite F, thioglycollate,
tetrathionate
▸ 3. Differential Media
▸ Allow the visualization of metabolic difference between groups of
bacteria.
▸ Examples: MAC, BAP, eosin methylene blue (EMB), and Hektoen
enteric agar (HEA).
▸ MAC differentiates lactose fermenters (pink colonies) from non-
lactose fermenters (colorless colonies).
▸ BAP differentiates hemolytic patterns of streptococci
▸ Alpha hemolysis
▸ Partial lysis and greenish discoloration around colony
▸ Streptococcus pneumonia, S. mitis, S. mutans, S.
salivarius
▸ Beta hemolysis
▸ Complete lysis of RBCs around the colony
▸ Streptococcus pyogenes and Streptococcus
agalactiae
▸ Gamma hemolysis
▸ There is no lysis of RBCs and no apparent change of
agar around the colony
▸ Streptococcus bovis
▸ Neutral red is the pH indicator in MAC that detects the fermentation
of sugar and eventually the production of acid.
▸ 4. Selective Media
▸ Incorporated with antibiotics, dyes, or chemicals to inhibit the
growth of other organisms while promoting the growth of desired
organism.
▸ Examples: Salmonella Shigella Agar (selective for Salmonella and
Shigella), Thiosulfate Citrate Bile Salt Agar (Selective and
Differential for Vibrios), Thayer Martin Agar (inhibits yeast)
B. Inoculation of Specimen
▸ STREAKING is the most common manner of inoculation.
▸ A specimen that is collected through a swab is inoculated by gently rolling the tip
of the swab onto the upper portion of the plate; the inoculated area should be
streaked by a sterile loop afterwards.
▸ The placement of fluid specimens or swabs into broth or liquid media.
▸ Stabbing technique is used in semi-solid/ solid mediums in tubes (inoculating
needle)
▸ Overlapping inoculation is used for antimicrobial sensitivity test or disk diffusion
method which is done on Mueller Hinton Agar (MHA).
C. Bacterial Growth Curve
▸ The generation time of bacteria in a culture can be as brief as 20 minutes for a
fast growing bacterium such as Escherichia coli or as long as 24 hours for a slow
growing bacterium such as Mycobacterium tuberculosis.
▸ Stages of Bacterial Growth
▸ 1. Lag Phase or the Period of Rejuvenescence
▸ When there is no cell division or an abrupt increase in the cell
number.
▸ The start of biosynthesis although there is no increase in cell mass.
▸ Adjustment phase to a new environment.
▸ 2. Log or Exponential Phase (Balance Growth)
▸ When microorganisms are actively growing and dividing.
▸ The bacteria increase logarithmically since cellular production is
most active during this period.
▸ It is the phase in which microorganisms are utilized in physiological
and biochemical testing.
▸ 3. Stationary/Plateau Phase
▸ When there is a balance between cell division and dying
organisms, although the number of viable microorganisms remains
constant.
▸ Metabolic activities of surviving cells slow down and nutrients are
becoming limited.
▸ Dead debris starts to accumulate.
▸ 4. Death or Decline Phase
▸ When there is a cessation of bacterial growth as the number of
dead cells exceeds the number of living microorganisms.
▸ There is a loss of nutrients and increase in the amount of toxic
waste.
III. Assignment
Draw the procedure for Gram staining and AFB (Kinyoun method) staining. Take a
picture and place the picture on Word.
IV. Generalization/ Summarization
Give a summary or generalization of what you have learned from Lesson 2: Bacterial
Identification and Processing. Minimum of 5 sentences.
V. Assessment Quiz with Answers
Choose the best answer among the choices.
1. This is the increase of bacteria in blood causing harm to the patient.
Bacteremia
Pathogenic Infection
Nosocomial Infection
Septicemia
2. Which type of microscope allows the detailed examination of internal structures in
living organisms?
Brightfield microscope
Phase-contrast microscope
Dark field microscope
Electron microscope
3. In which stage are bacteria utilized for antimicrobial testing?
Plateau phase
Decline phase
Exponential phase
Stationary phase
4. Which of the following culture media is used to differentiate the hemolytic patterns of
streptococci?
Blood agar plate
Chocolate agar plate
Nutrient agar
MacConkey agar
5. What kind of bacteria retains the crystal violet- iodine complex dye and purple color
after decolorization?
Gram-positive
Gram-negative
Non acid-fast
Acid-fast
6. What is the most common pathogen of the throat and nasopharynx?
Streptococcus salivarius
Streptococcus agalactiae
Streptococcus pyogenes
Streptococcus viridans
7. What term refers to the time required for the bacteria to double its population?
Proliferation time
Growth period
Generation time
Period of rejuvenescence
8. Which type of staining technique is aimed at coloring the forms and shapes of the
cells?
Gram staining
Simple staining
Negative staining
Differential staining
9. Most common cause of UTI in young sexually active females.
Streptococcus pyogenes
Escherichia coli
Staphylococcus saprophyticus
Staphylococcus aureus
10. In Gram staining, what happens to the organism if the decolorizing agent is
insufficiently applied?
The organism may appear as falsely Gram-positive cells.
The organism will exhibit red color
The Gram-positive complex will be removed.
The organism will be unstained.
11. In which microscope can Treponema pallidum be visibly observed?
Brightfield microscope
Electron microscope
Phase-contrast microscope
Dark field microscope
12. Which of the following is not a function of cedar wood oil in microscopy?
It is used to fill the space between the objective lens and the glass slide.
It enhances the resolution of the microscope.
It stains the specimen to improve visualization.
It prevents light rays from dispersing and changing wavelength after passing
through the samples.
13. What is the normal flora of the vaginal area during childbearing age?
Corynebacterium
Streptococci
Lactobacillus
Staphylococci
14. The sputum specimen is gram stained and observed under the microscope. It was
found to have 9 squamous epithelial cells and 26 PMNs/lpf. Should the specimen be
rejected?
Yes
No
15. Which type of microscope can visualize objects that are smaller than 0.2 μm using a
100,000x magnification?
Electron microscope
Dark field microscope
Brightfield microscope
Phase- contrast microscope
16. Which staining method is utilized to demonstrate the presence of diffuse capsule
surrounding the bacteria?
Simple staining
Gram staining
Negative staining
Acid-fast staining
17. Which kind of medium differentiates the fermentation of lactose among different
bacteria?
Columbia Agar
Thayer-Martin agar
Triple sugar iron agar
MacConkey agar
18. After acid- fast staining, what color will an acid- fast bacteria have when observed
under the microscope?
violet
pink
blue
green
19. All of these bacilli are Gram-positive except:
Nocardia
Clostridium
Bacillus
Brucella
20. What is the recommended enrichment broth for the isolation of Vibrio species?
Alkaline peptone broth
Selenite F
Tetrathionate broth
Lim broth
VI. References
▸ Mahon, C.R., Lehman, D.C., Manuselis, G. (2014). Textbook of
Diagnostic Microbiology (5th ed.). New York: Saunders
▸ Bailey, W. R., Scott, E. G., Finegold, S. M., & Baron, E. J. (1986). Bailey and Scott's
Diagnostic microbiology. St. Louis: Mosby.
▸ Rodriguez, M.T. (2018). Review Handbook in Diagnostic Bacteriology. C&E
Publishing Inc.
