ALYSSA E.
AMPER – BMLS 3D
ANTIMICROBIAL SUSCEPTIBILITY TESTING
Last step for work up in microbiology
laboratory
To know if a panel of antibiotics is specific
for that organism is susceptible or resistant.
DISK DIFFUSION METHOD (KIRBY-BAUER TEST)
For Aerobes and Facultative microorganisms
Mueller Hinton Agar (MHA) – culture
medium for antimicrobial susceptibility
testing; it is non selective; no inhibitory
agents present
Standard: 0.5 McFarland/Barium Sulfate
Suspension (99.5 mL of 1% H2SO4 and 0.5
mL of 1.175% BaCl2)
Standard inoculums: 1.5 x 108
organisms/ml
pH: 7.2-7.4 (important factor; too acidic or
too alkaline organism will die)
Depth: 4mm
Condition: Aerobic, No CO2
Temperature: 35-37C
Incubation time: 16-18 hours
Antibiotic disc: 6mm (standard size)
ANTIMICROBIAL SUSCEPTIBILITY TESTING
15mm: Disk distance (para di mag sapaw
ang zone of inhibition)
150mm (size/diameter of petri dish) = 12
discs (kadaghan sa antibiotic ibutang)
100mm = 5-6 discs
BACTERIOLOGY-FINALS
False Resistant False Sensitive
15 mins (delay of disc 15 mins (delay of
application) incubation)
Increase moisture Increase drying
Thick medium Thin medium
Increase organisms = Decrease organisms =
Decrease ZOI Increase ZOI
PROCEDURE
1. Pure inocula are obtained by selecting 4-5
colonies of the same morphology
2. Colonies are suspended into 0.85% NSS to
achieve a turbid suspension
3. Bacterial suspension & McFarland solution
are compared by matching the turbidity if the
tubes against a dark background
4. Turn the plate 60 degrees between each
streaking (Overlapping Streaking)
5. Within 15 mins of inoculation, antimicrobial
agents are applies onto MHA
6. Within 15mins of disk placement, plates are
inverted and incubated at 35 C for 16-18
hours.
7. Diameter of each inhibition zone is
measured using a caliper or ruler.
READING OF PLATES AND INTERPRETATION OF
RESULTS
The lawn must be confluent or almost
confluent – if the growth is poor and
nonconfluent, the test should be repeated
Provided growth is satisfactory, the diameter
of each inhibition zone is measured using
caliper or ruler
ALYSSA E. AMPER – BMLS 3D BACTERIOLOGY-FINALS

Appearance of individual colonies is

unacceptable.

Calliper

Susceptible: microorganism should respond

to therapy with that antimicrobial agent
Intermediate: microorganism falls into a
concentration )
range of susceptibility in which the MIC ( minium inhibitory
approaches or exceed the level of
antimicrobial agent that can be achieved and
for which clinical response is likely to be less
than with a susceptible strain
Resistant: no zone or small zone of
inhibition – antibiotic is not the appropriate
choice for treatment
ALYSSA E. AMPER – BMLS 3D
Mycobacteria
GENERAL CHARACTERISTICS
Gram staining (possible): faintly purple to
blue
The cell wall has extremely high lipid
(Mycolic Acid) content
Mycolic acid - resist decolorization with acid-
alcohol; major component of the cell wall of
mycobacterium
o 60% of the structure of
mycobacterium is a mycolic acid
composition
o Acid fast bacilli (AFB): basic
distinguishing feature; reporting
Most pathogenic species requires 2-6 weeks
of incubation
Most are slow growers except
Mycobacterium fortuitum-chelonei: 3 days
on Modified MaConkey agar
Mycobacterium leprae – fails to grow in
vitro (using agarose medium); cause leprosy;
siya rajud isa ang dili mutubo
Mycobacterium tuberculosis
Also known as Koch’s bacillus/Tubercle
Bacilli
MTB was first described by Robert Koch in
1882
Agent of Tuberculosis
o A chronic infection (host carry the
organism chronically)
o 20th century – 2nd leading cause of
death
Transmitted by the means of aerosol
TUBERCULOSIS
A disease of the respiratory tract (Pulmonary
TB (PTB))
Acquired by person with active disease by
means of:
1. Coughing
2. Sneezing or talking
BACTERIOLOGY-FINALS
Fibrous scars (lungs) – regression and
healing of the primary lesion
PRIMARY TUBERCULOSIS
PATHOGENESIS
1. M. tuberculosis cells are phagocytized by
alveolar macrophages and are capable of
intracellular multiplication
2. In a person with adequate cellular immunity,
T cells arrive within 4 to 6 weeks owing to
macrophage-activating polypeptides termed
lymphokines
3. This enables the macrophage in the area of
infection to destroy the intracellular
mycobacteria
4. There is then a regression and healing of the
primary lesion and any disseminated foci of
infection by the M. tuberculosis organisms.
In many expose individuals, the immune
system does not eliminate the bacteria
If there is little antigen and a strong
hypersensitivity reaction, a hard tubercle or
granuloma may be formed – an organization
of lymphocyte, macrophages, fibroblasts,
and capillaries.
With granuloma formation, healing occurs,
as well as fibrosis, encapsulation, and
calcification, with scar formation as a
reminder of the past infection
o Any scars observe on the chest x-
ray – indicates past infection
POTT’S DISEASE
AKA: “Tuberculosis Spondylitis” or
Skelertal TB of the Spine
Invasion into the spinal vertebrae (graveform
of spine)
MILIARY TUBERCULOSIS
Seeding of many organs outside the
pulmonary tree (Extrapulmonary
Tuberculosis)
ALYSSA E. AMPER – BMLS 3D BACTERIOLOGY-FINALS

Common sites: spleen, liver, lung, bone TUBERCULIN TEST: Method of Administration
marrow, kidney, adrenal gland and eyes
(chronic) Mantoux test: intracutaneous injection
The standard dose 0.1 ml of Purified Protein
TREATMENT Derivative (PPD) or 5TU (tuberculin units)
(+) erythema and hardening of the site of
First-line TB drugs – drugs administered injection (10mm)
first if you have the disease
o Rifampicin, Isoniazid, OTHER METHODS OF ADMINISTRATION
Pyrazinamide, Ethambutol,
Streptomycin (RIPES) Von pirquet test
Second-line agents – use this drugs if the Vollmer patch test
body is resistant to the first-line of drugs Moro percutaneous test
o Ethionamide, Capreomycin, Tuberculin tine test
Ciprofloxacin, Cycloserine,
LABORATORY DIAGNOSIS
Rifabutin, Ofloxacin, Kanamycin
MDR-TB (Multi drug resistance- Sputum and bronchial aspirates are the most
Tuberculosis) common : 2 sputum samples (early-morning
o Resistant ot Rifampicin and specimens that are collected 1 hour apart or
Isoniazid at least within three consecutive days
XDR-TB (extremely drug resistant- Purulent
Mycobacterium tuberculosis) Mucoid
o MDR-TB + resistant to 2nd-line TB Salivary – pwede ma reject na specimen
drugs and Fluoroquinolone Blood-stained
Coiling of sputum
PREVENTION

Bacillus Calmette-Guerin (BCG) vaccine

is prepared from a strain of the attenuated
(virulence-reduced) live bovine tuberculosis
bacillus, Mycobacterium bovis, that has
lost its ability to cause disease in humans.

TUBERCULIN TEST

Skin test which identifies tuberculosis

ACCEPTABLE SPECIMENS FOR PROCESSING
infection, recent or past, with or without
disease. Persons infected with tubercle RESPIRATORY SPECIMENS
bacilli develop hypersensitivity to the
proteins of the organisms. Spontaneously expectorated sputum
o Red – recent or past infection Normal saline-nebulized, induced sputum
o AKA: Mantoux test Transtracheal aspirate
Bronchoalveolar lavage
Bronchoalveolar brushing
Laryngeal swab
Nasopharyngeal swab
ALYSSA E. AMPER – BMLS 3D BACTERIOLOGY-FINALS

BODY FLUIDS i. Benzalkonium chloride –

shortens the exposure
Pleural fluid
time
Pericardial fluid
Joint aspirate
Gastric aspirate
Peritoneal fluid LABORATORY DIAGNOSIS
Cerebrospinal fluid
Stool Ziehl-Neelsen and Kinyoun stains
Urine
Pus BOTH uses:

BODY TISSUES Carbolfuchsin as the primary stain

Acid-alcohol as a decolorizing agent
Blood Methylene blue counterstain
Bone marrow biopsy/aspirate
Ziehl-neelsen: hot stain - heat as mordant
Solid organ
Kinyoun: cold stain - turgitol as amordant
Lymph node
Bone
Skin

DECONTAMINATION AND DIGESTION

To isolate acid fast organisms

Sample submitted is too viscous (intact), add Direct Sputum Smear Microscopy
mucolytic agents
DIGESTION –to liquefy to release acid fast Smear size: 2x3 cm
bacilli and expose normal flora for Read at 300 fields under OIO
decontamination. o Cedarwood oil
DECONTAMINATION – process the toxic Gram stain qualifies specimen if acceptable
agents use to kill rapidly growing normal Battlement method – count
flora. Barlett’s criteria
To isolate acid fast organisms o <10 epithelial cells
1. Sodium Hydroxide (NaOH): usual o >25 PMNs (polymorphonuclear
concentration 2-4%; serves as a digestant cells)
(mucolytic) and decontaminating agent
NATIONAL STANDARD REPORTING SCALE
2. N-Acetyl-L-Cysteine (dithiothreitol) as
Report Number of organism
mucolytic agent with 4% NaOH seen
(decontaminating agent): recommended 0 No AFB seen in 300
technique fields
3. Zephiran (benzalkonium chloride) + +n 1-9 AFB/ 100 fields
trisodium phosphate (Z-TSP) 1+ 10-99 AFB/100 fields
a. TSP liquefies sputum rapidly but 2+ 1-10 AFB/in at least 50
requires a long exposure time to fields
3+ More than 10 AFB/ in at
decontaminate
least 20 fields
ALYSSA E. AMPER – BMLS 3D
CULTURE and CULTURE MEDIA
Agar-based (serum-albumin agar media)
1. Middlebrook 7H11: 0.1% casein
hydrolysate, improves recovery of isoniazid-
resistant MTB
2. Mitchison’s selective 7H11
Liquid Media
Mycobacterium spp. grow more rapidly in
liquid medium, and it can be used for both
primary isolation and subculturing
1. Bactec 12B medium
2. Middlebrook 7H9 broth: for aseptically
collected specimens from normally sterile
sites
3. Middlebrook 7H13: for larger volumes of
bone marrow or blood specimens
Egg base
With egg yolk (lipid source) and malachite
green to inhibit the growth of contaminats
1. Petragnani: highest concentration of
malachite green
2. Lowenstein-Jensen: most commonly used
3. American Thoracic society (ATS) medium
CULTURE
Colonies of this slowly growing species are
typically raised, with a dry, rough
appearance. The colonies are nonpigmented
and classically described as being buff-
colored.
On Lowenstein-Jenson (LJ) slants, M.
tuberculosis typically has a crumbly, “bread-
crumb” appearance, whereas M. avium
complex has a bright yellow, smooth,
characteristic colony (so-called “condom
colony”).
These morphologic features are useful in
making a presumptive speciation, which is
usually confirmed by DNA probe.
BACTERIOLOGY-FINALS
BIOCHEMICAL TESTS
NIACIN ACCUMULATION
Most mycobacteria possess the enzyme that
converts free niacin to niacin
ribonucleotide. However, 95% of M.
tuberculosis isolates produce free niacin
(nicotinic acid) because this species lacks
the niacin-connecting enzyme
NIACIN + Cyanogen bromide (reagent) + AMINE =
yellow-pigmented compound
NITRATE REDUCTION
The production of nitroreductase, which
catalyzes the reduction of nitrate to nitrite, is
relatively uncommon among mycobacterium
spp., but a positive result may be seen in M.
kansasii, M. szulgai, M. fortuitum, and M.
tuberculosis.
CATALASE TESTS
Mycobacteria are catalase positive.
However, not all strains produce a positive
reaction after the culture is heated to 68 C
for 20 minutes (heat-stable catalase)
Most M. tuberculosis complex organisms do
not produce heat-stable catalase
Semi-quantitation of catalse production
o Tween 80 (a detergent) + 30%
H2O2 + 2-week-old culture grown in
an agar deep
The reaction is read after 5
minutes
The column size is
recorded as greater than
or less than 45 mm.
ALYSSA E. AMPER – BMLS 3D BACTERIOLOGY-FINALS

TWEEN 80 HYDROLYSIS Mycobacterium bovis

Some mycobacteria possess a lipase that
can split the detergent Tween 80 into oleic
acid and polyoxyethylated sorbitol
1. The pH indicator, neutral red, is initially
bound to Tween 80 and has an amber color
2. After hydrolysis of Tween 80, neutral red can
no longer bind, and it is released, causing a
pink color to form.
POSITIVE: Pink
NEGATIVE: Yellow
ARYSULFATASE TEST
Produces TB primarily in cattle but also in
other ruminants, as well as in dogs, cats,
swine, parrots, and humans
Most strains of M. bovis are niacin-negative
Mycobacterium leprae
The causative agent of Hansen disease
(leprosy), an infection of the skin, mucous
membranes, and peripheral nerves.
Hansen disease is not considered highly
contagious

Principle: detects Rapid growers TWO MAJOR FORMS:

Positive result: Red/Pink
1. Tuberculoid leprosy: symptoms of
Strongly (+): M. fortuitum-chelonei tuberculoid leprosy include skin lesions and
Negative: MAC, M. tuberculosis nerve involvement (loss of sensation).
Biochemical + - Patients eventually exhibit an effective cell-
Tests mediated immune (CMI) response.
Niacin M. tuberculosis M. bovis (no 2. Lepromatous leprosy: Characterized by
Accumulation (yellow) color change) skin lesions and progressive, symmetric
Nitrate M. tuberculosis, No color change nerve damage and do not produce an
Reduction M. kansasii, M. effective CMI response (very poor prognosis)
szulgai, M.
fortuitum (red) LEPROMIN TEST
Catalase Test Most M. tuberculosis
mycobacteria (no bubbles, Skin test consisting of a heat-killed
(vigorous <45mm height of suspension of M. leprae prepared from
bubbling, >45mm bubbles) lepromatous nodules.
height of bubbles)
This test however lacks specificity.
Tween 80 Non- M. tuberculosis
Hydrolysis photochromogens (no color change) TYPES OF REACTION:
and o Early Fernandez reaction:
Scotochromogens induration appears in 24-48 hours
(pink) (redness/swelling)
Arylsulfatase M. fortuitum- MAC, M. o Late Mitsuda reaction: indurated
Test chelonei tuberculosis nodule develops after 3-4 weeks
(red/pink)
CULTIVATION

Non-culturable on artificial medium

Can be grown experimentally only in:

o Footpad of mice: success is in the
low temperature (30 C) of footpads
ALYSSA E. AMPER – BMLS 3D BACTERIOLOGY-FINALS

o Armadillos: animals with low body

temperature

RUNYON’S CLASSIFICATION

Photochromogens (Group I) – pigmented

only when exposed to light
M. kansasii: “yellow bacillus”
M. marinum: means “of the sea”, agent of
swimming pool granuloma

Scotochromogens (Group II) – pigmented

both in darkness and light
M. xenopi: branching colonies with aeriel
hyphae on cornmeal agar described as
“bird’s nest”
M. gordonae: “tap water” bacillus

Nonphotochromogenic (Group III) –

nonpigmented both in light and darkness
M. avium – M. intracellulare (Batter bacillus):
complex-opportunistic pathogens in patients
with AIDS and other immune dysfunctions
M. terrae: “radish bacillus”

Rapid growers (Group IV) – Produce

visible growth within less than 7 days
M. fortuitum-chelonei complex: grows in Mac
without crystal violet (modified MAC)