Bacteriology

Bachelor of Science in Medical Laboratory Science

Lecture-based/PPT-based/Book-based
Mr. Kristan J. Dela Cruz RMT, DTA, MLS (ASCPi) CM

g) Listeria
TOPIC OUTLINE h) Nocardia
i) Actinomyces
I. Staining Techniques
II. Cultural Method III. All spirochete organisms are reported as gram (-).
III. Serological IV. Yeasts (fungi) are reported as gram (+)
IV. Biochemical Methods V. Microorganism that does not need gram stain;
V. Bacterial DNA Testing a) Rickettsia/Chlamydia – intracellular
VI. Anti-susceptibility Testing b) Mycoplasma and Ureaplasma – no cell wall
c) Spirochete – can’t resolve by Bright Field
Staining Microscope.
d) Legionella and Spirochetes – silver
 Stains are organic or aqueous preparations of dyes that
impregnation technique is most useful.
impart a variety of colors to tissue or microorganisms.
 Stains are classified as either simple or differential. Simple
stains impart the same color to all structures, whereas
differential stains contain more than one dye and impart
different colors to various structures.
 In Simple staining, a single stain is used. The examples of
this are malachite green, methylene blue, crystal violet,
calbolfuchsin, safranin.
 Differential staining divides bacteria into groups. Example
of this are gram staining and Acid-fast staining (AFB).
- It has 4 components:
1. Primary stain
2. Mordant
3. Decolorizer Acid Fast Stain
4. Counter Stain
TYPES OF STAINING METHODS

Methylene Blue
 In the methylene blue stain, the specimen can be observed
for the presence of microorganisms as well as for their size,
shape, and morphology

Gram Stain
 The Gram stain will differentiate gram-positive bacteria
from gram-negative bacteria. The method, first introduced by
Hans Christian Gram in the late 1800s, has been modified and
adjusted numerous times. - Acid-fast stains are used to stain mycobacteria that
General Rule in Gram Staining possess thick, waxy cell walls. Once stained, these
bacteria resist decolorizing by acid alcohol, and thus
the designation acid fast.
- Stains the myolic acid in the cell wall of bacteria.
- There are 2 types of AFS:
 Zeihl-Neelsen – hot method; employs heat to dry
the stain

I. All cocci are gram (+) except NVM

a) Neisseria
b) Veilonella
c) Moraxella
II. All bacilli are gram (-) except MCCBELLNA
a) Mycobacteria
b) Corynebacteria
c) Clostridia
d) Bacillus
e) Erysiphilotrix
f) Lactobacillus

TRANSCRIBED BY: BSMT 2YB-1A Group 1 1

Bacteriology
Bachelor of Science in Medical Laboratory Science
Lecture-based/PPT-based/Book-based
Mr. Kristan J. Dela Cruz RMT, DTA, MLS (ASCPi) CM

 Kinyoun – cold method; employs Tergitol

Objective of Staining

Carbolfuchsin 1. To appreciate the appearance and morphology of

bacteria.
Tergitol
2. To differentiate one group of organism from another
Acid alcohol
group of organisms.
(3% HCl in 95% 3. To identify an organism via staining the special
Ethanol)
structure of the organism.
Methylene
blue Principle of Staining

 Chromophores are the colored portion of the dye

Fluorescent Stains
There are three fluorescent stains we used in order Acidic dyes
to stain different types of microorganisms, and these - anionic chromophores
are: - anionic or possess negatively charged groups that
1. Rhodamine-auramine is a fluorescent stain used binds to positively charged cell structures.
to stain mycobacteria. The stains bind to the mycolic - E.g. eosin, rose Bengal, and acid fuchsin
acid in the organism’s cell wall, staining the bacteria Basic dyes
yellow to orange.
- cationic chromophores
2. Acridine orange selectively binds to any nucleic
- these are the common dyes used in bacteriology.
acid and is useful in demonstrating small amounts of
bacteria in blood cultures, cerebrospinal fluid, - cationic or have positively charged groups that
urethral smears, and other exudates. binds to negatively charged molecules.
3. Calcofluor white is another fluorescent stain used - E.g. methylene blue, basic fuchsin, crystal violet,
to stain specimens directly for the presence of fungi. safranin and malachite green
The fungal elements appear blue-white when
observed with a fluorescent microscope. Cultural Methods
Fungal Stains  Culture media
- Lactophenol cotton blue, methenamine silver, and - Culture media transport systems are preferred over swab
periodic acid-Schiff (PAS) also can be used to stain system transport systems. Culture media systems include the
fungal mycelium. The PAS stain is generally preferred JEMBEC plate
for staining direct clinical material. - it is also an artificial substance or material in which organism
Antibody-Conjugated Stains finds nourishment and can reproduce.
- By binding specific antibodies to stain, one can
detect the corresponding organism. Specific
applications include:
1. Fluorescein-conjugated stains. Antibodies are
bound to fluorescein isothiocyanate, which binds to
the specific antigen if it is present. Examples include
the direct fluorescent antibody (DFA) technique
used to directly detect Legionella pneumophila and
Bordetella pertussis in clinical specimens.
 Culture: microorganisms in specimen can be cultivated and
2. Enzyme-conjugated stains. Enzymes such as
grown in artificial media
horseradish peroxidase are bound to a specific
 Culture medium: contains nutritional requirements
antibody.
needed for bacterial growth
Preparation of Smear
 There are 3 types of medium:
 The first step in staining is the preparation of a 1. Pure culture - one species
smear. 2. Mixed culture – more than one species
 Smears are prepared by rolling a swab on a 3. Stock culture – for American Type Culture Collection
(ATCC)
microscopic slide and placing a drop of liquid broth
According to Consistency
or specimen on a microscopic slide.
 Agar
 Smears are allowed to air dry or are dried using a
- usually derived from red algae, solidifies at 40-to
slide warmer. 50°C and melts at 80-90°C.
- cooling temperature for distribution of media to
plate 55-60°C; amount 20-25ml per plate.

A. Liquid broth media

 Nutrients are dissolved in water, and bacterial growth is
indicated by a change in broth’s appearance from clear to
turbid (i.e. cloudy)
 Nutrient Broth
 Brain Heart Infusion
 Alkaline Peptone Water
 Thioglycolate broth

TRANSCRIBED BY: BSMT 2YB-1A Group 1 2

Bacteriology
Bachelor of Science in Medical Laboratory Science
Lecture-based/PPT-based/Book-based
Mr. Kristan J. Dela Cruz RMT, DTA, MLS (ASCPi) CM

B. Solid Agar Media  The laboratory performs direct stains, plates the specimen
 2-3% Agar on appropriate culture media, and incubates the plates at the
suitable temperature and atmosphere. The plates are
 Made by adding a solidifying agent (agarose) to the
examined and interpreted for the presence of pathogens,
nutrients and water.
most often at 24 and 48 hours.
 Liquefiable:
o Liquid > solid > liquid
o Ex. SSA, BAP, MAC
 Non-liquefiable:
o Will no longer liquefy when needed According to Functions
o Ex. Rice Medium (Fungi) A. Nutritive Media
 Contain nutrients that support growth of most
C. Semi Solid Media nonfastidious organisms without giving any particular
 For special purposes where agar is added to media in organism a growth advantage.
concentrations that are too low to solidify them.  Examples: Nutrient Agar, Tryptic Soy Agar, Sabouraud’s
Dextrose Agar for Fungi

B. Enrichment Media
 This media can be solid or liquid
 Contain specific nutrients (antibiotics or dyes) required for
the growth of particular bacterial pathogens that may be
present alone or with other bacterial species in a patient
specimen. (Bailey)
According to Composition  When a substance is added to a liquid medium which
A. Synthetic or Chemically Defined Media inhibits the growth of unwanted bacteria and favors the
 Composition is exactly known, qualitatively and growth of wanted bacteria. (Kumar)
quantitatively.

 They are chemically defined media prepared from pure Thioglycollate for the isolation of
anaerobes
chemical substances.
Tetrathionate broth inhibits coliforms while
 Examples: Simmons Citrate Agar, Mineral Glucose Medium allowing typhoid-
paratyphoid bacilli to grow
freely in fecal sample
Lysine, Indole, Motility for selective enrichment of
(LIM) group B Streptococci
Gram-negative (GN) broth selective enrichment of
enteric gram-negative
organisms
Selenite F (F for feces) is used for dysentery bacilli
broth
B. Non-synthetic or Complex Media Alkaline peptone water is used for Vibrio cholerae
 The medium has a known composition only with from faeces
approximation, the complex medium contains complex
ingredients, such as yeast extract or casein hydrolyzate, in C. Enriched Medium
unknown proportions  Enriched Media are prepared to meet the nutritional
 Example: Soy Agar, Nutrient Agar requirements of more exacting bacteria by the addition of
C. Tissue Culture substances such as blood, serum, or egg to a basal medium.
 From its name, it uses living tissue, cells or virus.  For fastidious microorganisms.
 In this culture, cytophatic effect is observed. Blood Agar Plate (BAP) is used for isolation of
 Examples: streptococci, pneumococci,
a) HeLA cells - cervical cancer cells. Haemophilus
b) A549 - lung carcinoma Chocolate Agar Plate (CAP) is used for isolation of
c) McCoy cells - for cultivation of Chlamydia neissseria (meningococci
and gonococci) and
trachomatis
Haemophilus
d) Vero cells - African green monkey kidney cells
Loeffler’s Serum Slope (LSS) is used for isolation of
e) Hep2 cells - laryngeal cancer cells
Corynebacterium
According to Formation dyptheriae
A. Tubed Buffered Charcoal-Yeast provides L-cysteine and
 Butt Extract Agar (BCYE) other nutrients required for
 Butt/Slant the growth of Legionella
 Slant pneumophilia
 Broth
B. Plated
D. Selective Media

TRANSCRIBED BY: BSMT 2YB-1A Group 1 3

Bacteriology
Bachelor of Science in Medical Laboratory Science
Lecture-based/PPT-based/Book-based
Mr. Kristan J. Dela Cruz RMT, DTA, MLS (ASCPi) CM

Contain one or more agents that are inhibitory to all - Medium: Enriched CAP
organisms except those “selected” by the specific growth - Inhibitors: antibiotics
condition or chemical.  Thayer-Martin
a) Deoxycholate Citrate Agar  Modified Thayer-Martin
b) Lowenstein-Jensen Medium  Martin-Lewis
c) Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS Agar)  New York City Agar
d) Phenylethyl alcohol (PEA) Agar with 5% sheep blood
e) Modified Theyer-Martin (MTM) Agar
Deoxycholate citrate agar this acts as a selective agent
(DCA) for dysentery bacilli
(isolation of Shigellae)
Lowenstein-Jensen Medium used for Mycobacterium
tuberculosis
Thiosulfate-citrate-bile salts Bile salt is a selective agent.
sucrose agar (TCBS) It feredurs the growth of
only Vibrio cholerae and
Selective Differential for Staphylococcus spp.
inhibits the growth of
Medium: Mannitol Salt Agar (MSA)
intestinal organisms.
Phenylethyl Alcohol (PEA) inhibits the growth of Inhibitor: 7.5% NaCl
with 5% sheep blood aerobic and facultatively Carbohydrate: Mannitol
anaerobic gram (-) rods and pH Indicator: Phenol Red
allows gram (+) cocci to  Acid – Yellow
grow  Alkaline – Red
Modified Thayer-Martin is an enrichment and MF: Yellow (S. aureus)
(MTM) agar selective medium for the Non-MF: Pink (S. epidermis and S. saphrophyticus)
isolation of Neisseria
gonorrhoeae, N.
meningitidis

 For Mycobacterium tuberculosis

- Cauliflower Colonies
- Medium: Lowenstein- Jensen
- Medium Inhibitor: Malachite Green

E. Differential Media
 Differential media provide distinct colonial appearances of
microorganisms to aid in their identification.
 Most differential media are used to isolate gram-negative
bacteria through the addition of ingredients that are
inhibitory for gram (+) bacteria.
 For Corynebacterium diphtheriae
1. MacConkey Agar (MAC)
- Jet Black Colonies
- MacConkey agar contains lactose, bile salt, neutral
- Medium: Mueller Tellurite
red indicator, and crystal violet. The bile salt and
- Medium Inhibitor: Potassium Tellurite
crystal violet inhibit the growth of gram-positive
bacteria.
- If the organism can ferment lactose, the colonies
appear pink to red in color.
- If the organism is unable to ferment lactose, the
colonies appear clear.
2. Hektoen Enteric (HE) agar
- contain agents that inhibit the growth of all
bacteria except those that are sought. These media
allow one to select for pathogens through the
 For gram stain positive bacteria inhibition of normal flora.
- Opaque colonies - contains lactose, sucrose, salicin, sodium
- Inhibitor: Phenylethyl alcohol (PEA) thiosulfate, ferric ammonium citrate, and
- Inhibits gram (-) bacteria bromthymol blue indicator.
- for the isolation and differentiation of Salmonella
and Shigella spp. from other gram (-) enteric bacilli.

 For N. gonorrhoeae

TRANSCRIBED BY: BSMT 2YB-1A Group 1 4

Bacteriology
Bachelor of Science in Medical Laboratory Science
Lecture-based/PPT-based/Book-based
Mr. Kristan J. Dela Cruz RMT, DTA, MLS (ASCPi) CM

G. Sugar Media & Transport Media

 Sugar media is used for identification of most organisms,
sugar fermentation reactions are carried out.
Example: Hiss Serum Sugars, TSI
 Transport medium is a holding medium designed to
preserve viability of microorganisms in the specimen but not
allow multiplication.
1. Stuart’s transport medium for Gonococci
2. Amies transport medium for Gonococci
3. Buffered glycerol saline for enteric bacilli

3. Xylone-lysine-deoxycholate (XLD) agar

- just like HE agar, XLD agar selectively inhibit gram-
positive bacteria and gram-negative coliform
bacteria and permit the isolation of
stool pathogens.
- for Shigella spp. and Salmonella spp.
H. Anaerobic Media
F. Indicator Media  These media are used to grow anaerobic organisms and
 These media contain an indicator which changes color contain reducing substances.
when a bacterium grows in them.  Anaerobic media contain supplements, including hemin,
Examples: blood, and vitamin K, as well as sodium bicarbonate, which
 Wilson and Blair medium (Bismuth Sulfite Agar) provides a source of CO2. Reducing agents, such as
o Bismuth sulfite and brilliant green agar are thioglycollic acid, sodium thioglycollate, and L-cystine, are
suitable for the isolation of Salmonella added to the media to absorb oxygen.
serotype Typhi.  Anaerobic media do not need to be stored anaerobically,
o There is incorporation of sulfute in Wilson they are held in a reduced state 8 to 16 hours prior to
and Blair medium. inoculation.
o S. typhi have black metallic sheen Examples:
1. Thioglycollate broth

 MacConkey Agar 2. Cooked Meat broth

o selects gram-negative bacteria and
differentiate lactose fermenters from
lactose non-fermenters.
o If the organism can ferment lactose, the
colonies appear pink to red in color.
o If the organism is unable to ferment
lactose, the colonies appear clear.

I. Antibiotic Susceptibility Testing

A. Mueller Hinton Agar
 suitable medium for testing the susceptibility of
microorganisms
 can be used to cultivate Neisseria.

TRANSCRIBED BY: BSMT 2YB-1A Group 1 5

Bacteriology
Bachelor of Science in Medical Laboratory Science
Lecture-based/PPT-based/Book-based
Mr. Kristan J. Dela Cruz RMT, DTA, MLS (ASCPi) CM

Gross Colony Characteristics

1. Hemolysis

 Alpha – partial hemolysis

 Beta – complete clearing
 Gamma – no hemolysis

2. Size – large, medium, pin-point

3. Form of Margin (Texture) – filamentous, irregular, smooth,

rough

8. Pigment

 Pseudomonas aeruginosa – green

 Serratia mercescens – red/orange
 Kluyvera – blue
 Staphylococcus aureus – white
 Micrococcus luteus – white

Hemolysis of Streptococci

9. Odor

 Staphylococcus aureus – old sock

 Pseudomona aeruginosa – fruity or grape-like
 Proteus mirabilis – putrid
 Haemophilus – musty basement
4. Elevation – flat, raised, umbonate, umbilicate, convex  Norcardia – freshly plowed field

Serological Tests
 Detection of antibodies in patient’s serum against specific
antigen

 ELISA, ASO, Western blot

 ELISA - In enzyme-linked immunosorbent assays

(ELISA), antibody or antigen is bound to an enzyme
that is able to catalyze a reaction. The antibody-
5. Density/Transparency – transparent, opaque, translucent, binding site remains free to react with antigen, and
opalescent the enzyme catalyst remains unaltered during the
6. Color – white, gray, yellow reaction. The colored end product is then measured
spectrophotometrically.
7. Consistency – brittle, creamy, sticky, dry  ASO - group A streptococcal infections can be
diagnosed through the detection of antistreptolysin
O, an antibody in the patient’s serum.
 Western blot - Very specific antibody proteins can be
detected using the Western blot technique. This is
very cumbersome techniques to perform in the
routine clinical microbiology laboratory. The
techniques are used in reference and research
laboratories. The confirmatory test for human
immunodeficiency virus (HIV).

Biochemical Methods
 Identification of bacterial species based on the differences
in the biochemical activities of different bacteria.

 Determines presence of bacterial enzyme

Bacterial DNA Testing

TRANSCRIBED BY: BSMT 2YB-1A Group 1 6

Bacteriology
Bachelor of Science in Medical Laboratory Science
Lecture-based/PPT-based/Book-based
Mr. Kristan J. Dela Cruz RMT, DTA, MLS (ASCPi) CM

 Test for DNA inside the gene  Broth volume: 0.05 – 0.1mL
 DNA probing and amplification (PCR)
 Schaedler’s broth, West Wilkins broth, BHI
 Sterile specimen
 Rapid (1 hour)
 Highly specific

How does PCR work?

 Five core ‘ingredients’ are required to set up a PCR.

1. DNA Template
2. Primers
3. DNA nucleotide bases also known as dNTPs
4. Taq polymerase
5. Buffer 2. Macrodilution

 The MIC and MBC of a particular antibiotic are determined

through serially diluting an anti

 Useful for laboratories testing a few antibiotics with a few

organisms.

 It uses test tubes

 Reference method for antibiotic susceptibility testing.

 Broth volume: >10mL

Antisusceptibility testing
 To detect the ability of antimicrobial agent to inhibit
bacterial growth in vitro

 To determine the susceptibility/resistance of organisms

against antimicrobial agent

 Minimum Bactericidal Concentration

o The lowest concentration of the antibiotic
that killed the bacterium
 Minimum Inhibitory Concentration
o The lowest antibiotic concentration that
inhibits in vitro visible growth.

A. Test Tube Dilution/Broth Dilution Method

B. Agar Dilution Method
 Involves challenging the organism of interest with
 The antimicrobial concentrations and organisms to be
antimicrobial agents in a broth environment (Mueller-Hinton
tested are brought together on an agar-based medium rather
broth)
than in a broth
 Specific amount of antibiotic is prepared in a decreasing  Standard inoculum size: 1x104 cfu/ml
concentration in broth by serial dilution techniques, and  Shelf life of agar dilution plate is only 1 week for most
standard numbers of the test organism is inoculated. antimicrobial agents.
 Reference method for testing anaerobes and N.
 Standard inoculum size: 5x105 cgu/ml gonorrhoeae
 Can be used to determine MIC and MLC concentrations.  Reference method for anaerobes: Brucella agar with laked
blood and vitamin K (Wadsworth method)

C. Disk Diffusion Method (Kirby-Bauer Test)

2 Types of Dilution Method
 Limited to aerobic and facultatively anaerobic bacteria.
1. Microdilution
 Filter paper disks impregnated with various antimicrobial
 Various concentrations of several antibiotics are tested agents of specific concentrations are carefully placed on an
against an inoculum. The MIC is determine using this method. agar plate previously inoculated with the bacterium being
tested
 It used multiwall microdilution trays  The Kirby-Bauer test is routinely used to determine the
susceptibility or resistance of a pathogenic organism to
various antimicrobial agents
 Standard inoculum size: 1.5x108 cfu/ml
 Susceptibility standard medium: Mueller Hinton Agar
(MHA)
 Results are reported as susceptible, intermediate, or
resistant

TRANSCRIBED BY: BSMT 2YB-1A Group 1 7

Bacteriology
Bachelor of Science in Medical Laboratory Science
Lecture-based/PPT-based/Book-based
Mr. Kristan J. Dela Cruz RMT, DTA, MLS (ASCPi) CM

 Principle: Based on the Inverse linear relationship 3. Heavy inoculum

between the diameter of the zone of inhibited growth 4. Thick medium
around the antibiotic disk and the logarithm of the MIC of 5. Prolonged incubation
the antibiotic False Sensitive
1. Delay of 15 minutes before incubation
2. Increase drying
3. Light inoculum
4. Thin Medium
5. Acidic pH affects the result of Tetracycline,
Novobiocin, Methicillin
6. Alkaline pH affects the result of Aminoglycocides,
Clindamycin, Erythromycin
Preparation of Pure Culture for Susceptibility Testing
7. Low temperature and old colonies (>1 day)
Preparation of Media
Antisusceptibility test cont…
 Media: Mueller Hinton Agar
D. D Test
 Depth of Agar: 4mm
 pH of Agar: 7.2 – 7.4 (Neutral)  To detect inducible clindamycin resistance among strains
 Filter paper disk/size: 6mm of S. aureus

Preparation of Inoculum  Uses if the result of clindamycin (susceptible) and

1. Subculture 4-5 colonies on TSB; incubate at 37°C for erythromycin (resistant) is different
3-5 hours
 (+) Result: Flattening or Blunting of Clindamycin to
2. Compare turbidity of subculture with McFarland
produce a “D” pattern
standard.
3. 0.5 McFarland Standard consists of 99.5ml of 1%  (-) Result: No flattening
Sulfuric acid and 0.06mL of 1.175% of Barium
Chloride
4. Equivalent of McFarland Standard: 1.5x108 cfu/ml
5. If turbidity is okay, inoculate on MHA; if too turbid
dilute using NSS or distilled water (overlap streaking)
6. Wait for 3-5 minutes before applying the disk

Plate Size and Number of Disks

 If plate size is 150 mm place no more than 12 E. E Test or Epsilometer Test

disks; if 100 mm place no more than 5-6 disks  Is a dilution test that uses a strip with single antibiotic of
 Distance of disk from center is 24mm, between 2 decreasing concentration along its length

 The strip is placed on the surface of the culture medium

inoculated with the desired organism and the antibiotic
diffuse into the surrounding agar

 For fastidious organism like Haemophilus spp.

 (+) Result: Ellipse of Growth of Inhibition (MIC)

disks is 15mm
 Inverts plates and incubate at 35-37°C; incubation
time is 16-18 hours; aerobic incubation NO CO2
 Measure zone of inhibition using RULER or
CALIPER and interpret if S, R, or I
 For media with blood measure from the top with
cover removed

F. Automated System – VITEK

 Advantages: High Sensitivity, Reduce TAT, Replaces
manual procedure

 Vitek 1 and 2
 Vitek-MS
 MALDI-TOF
 MicroScan WalkAway system
 BD Phoenix – nephelometry
Factors Affecting Zone of Inhibition
 BacT/Alert – culture system only
False Resistance
1. Delay of 15 minutes before disc application
2. Increase moisture

TRANSCRIBED BY: BSMT 2YB-1A Group 1 8