LOEFFLER’S ALKALINE METHYLENE BLUE

GRAM POSITIVE BACILLI (LAMB), E.G., ALBERT’S STAINING

GRAM + BACILLI LOEFFLER’S ALKALINE

● 2 subclass: METHYLENE BLUE (LAMB)
○ Spore-forming ● Pleomorphic beaded rods
■ Clostridium ● Reddish purple
■ Bacillus metachromatic granules
○ Non spore-forming or bars or polyphosphates
■ Corynebacterium diphtheria are apparent
■ Erysipelothrix rhusiopathiae ● Granules present:
■ Listeria monocytogenes ○ Babe’s Ernst
granules
(Metachromatic granules or Volutin)
■ For energy and food storage
● Methylene blue stain prepared from colonies
growing on Loeffler’s medium
● C. diphtheriae:
○ Diphtheroidal arrangement of bacilli with
dark Metachromatic granules
● Albert stain→ bluish-black
● Situated at the polar body
LABORATORY DIAGNOSIS:
CORYNEBACTERIUM DIPHTHERIAE C. ULCERANS & C. PSEUDOTUBERCULOSIS

● Resemble C. diphtheria
SPECIMENS ○ Producing distinctive, club-shaped,
diphtheroidal cells containing
metachromatic granules
SPECIMENS
● Swabs from the oropharynx (THROAT)
○ If a pseudomembrane is present:
■ Swab specimens from beneath
the membrane should be
collected
● Swab from nasopharynx (NOSE), or from
cutaneous lesions (SKIN)

DIPHTHEROIDS

● Mimic C. diphtheriae

I. MICROSCOPY II. CULTURAL METHOD

GRAM STAINING CULTURAL CHARACTERISTICS OF C. DIPHTHERIAE

● Aerobe and facultative anaerobe
GRAM STAINING ● Optimum T: 37 degrees C
● Pleomorphic gram-positive rods ● Optimum pH: 7.2
● Occur in angular arrangements ● Can grow on ordinary Nutrient agar but growth is
○ Chinese letters or Palisades improved by the presence of animal proteins
● Ends may be swollen→ club shape (blood or serum)
● BAM & Loefller’s→ 2 medium for C. diphtheriae

A. CULTURE MEDIA

5% SHEEP BAM, COLUMBIA CNA AGAR

5% SHEEP BAM, COLUMBIA CNA AGAR

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 FINALS | EXERCISE 22: GRAM POSITIVE NON SPORE-FORMING BACILLI

● Used to screen and rule out group A B-hemolytic ● Stimulates the growth of distinctive, club-shaped,
streptococci from throat infection diphtheroidal cells of C. diphtheriae and the
production of metachromatic granules in the
cells
● Poached egg colonies→ show no characteristic
differential features to distinguish from other
gram + bacilli

MEDIA CONTAINING CYSTINE & POTASSIUM

TELLURITE- SELECTIVE & DIFFERENTIAL FOR
CORYNEBACTERIUM SPP.

A. TINSDALE AGAR (TIN)

● Aka Cystine-sodium
thiosulfate-tellurite medium
● High concentration of
potassium tellurite:
○ Inhibitory to most
upper respiratory
tract normal flora
(other than
Corynebacterium
Species) majority of gram- negative
bacteria
○ Organisms capable of growing on the
medium are differentiated based on the
tellurite reductase activity→ resulting in
the reduction to tellurium, thus will grow
as black colonies.
● Bovine serum and horse serum:
○ Provide essential growth factors
● Sodium thiosulfate→ Provide sulfur for H2S
production PAI MEDIUM
● Cystine→ detects cystinase activity producing
brown halos around the colonies PAI MEDIUM
● MODIFIED TINSDALE ● Contains coagulated egg in:
○ Black colonies with dark brown halos ○ Distilled water
○ Inhibits upper respiratory tract flora ○ Glycerin

B. INOCULATION & INCUBATION

INOCULATION & INCUBATION

● Specimens should be inoculated to a blood agar
and a selective medium such as:
○ Tellurite place
■ CTBA
■ TIN
● Incubate at either in:
○ Ambient air
B. CYSTINE-TELLURITE BLOOD AGAR ○ 5% to 10% CO2 for 24-48 hrs
(CTBA)
● Heat infusion agar
supplemented with: C. COLONIAL CHARACTERIZATION
○ 5% rabbit blood
○ Tellurite C. DIPHTHERIAE
○ L-cystine
● Selective and differential
medium for C. DIPHTHERIAE
Corynebacterium spp. ● Appear as four distinct colony types (biotypes)
Similar to TIN designated:
● Black colonies ○ Gravis
● Inhibit other normal flora in upper respiratory ○ Mitis
tract ○ Intermedius
○ Belfanti
● These biotypes differ slightly in:
LOEFFLER’S SERUM MEDIUM ○ Gram’s stain morphology
○ Certain biochemical reactions
LOEFFLER’S SERUM MEDIUM ○ Historically: In the severity of the disease
● Poached egg colonies processed they produce
● Enhances granule
formation & pleomorphism
● Not support pneumococci
ON BAM
● Contains egg and beef
serum, rather than aagr→ ON BAM
to coagulate to produce a ● Produce colonies ranging from:
solid medium Small, Gray & Translucent (biotype

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 FINALS | EXERCISE 22: GRAM POSITIVE NON SPORE-FORMING BACILLI

intermedius) to Medium, White, and Opaque

(biotypes mitis, belfanti, gravis)
● C. diphtheria biotype mitis→ B-hemolytic

4 BIOTYPES

MITIS Regular edge (mild)

BELFANTI B-hemolytic

GRAVIS Most serious

INTERMEDIUS Intermediate
severity

ON CTBA

ON CTBA
● Cystine-Tellurite blood agar
● Incubated at 35 degrees C in a CO2
enriched atmosphere
● Colonies of C. diphtheria appear:
○ Black or gray, while most of
the usual oral flora are
inhibited D. TOXIGENICITY TEST

TOXIGENICITY TEST
● Once an organism is biochemically identified as a
possible C. diphtheriae, the isolate must be
tested for the ability to produce diphtheria toxin.

IN VITRO: ELEK TEST

ELEK TEST
● In vitro
● For demonstration of toxin production by C.
diphtheria
● Agar: Rabbit serum agar

● A filter paper strip impregnated with diphtheria

antitoxin is buried just beneath the surface of a rabbit
serum agar plate before the agar hardens.
● Strains to be tested and known positive and negative
toxigenic strains are streaked on the agar’s surface in a
line across the plate and at a right angle to the
antitoxin paper strip.
ON TIN ● After 24-48 hours of incubation at 37°C (or 35 degrees C
based sa ppt ni ma’am Krissa):
○ Plates are examined with transmitted light for
ON TIN the presence of fine precipitin lines at a
● All four biotypes of C. diphtheria grow as black 45-degree angle to the streaks.
colonies surrounded by dark brown halos ○ Presence of precipitin lines:
■ Strain produced toxin that reacted
with the homologous antitoxin.

Line 1 is the negative control.

Line 2 is the positive control.
Line 3 is a test organism that is a non toxigenic strain.
Line 4 is a test organism that is a toxigenic strain.

C. ULCERANS & C. PSEUDOTUBERCULOSIS

● Resemble C. diphtheriae
○ Producing black colonies surrounded by
brown halos on TIN

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 FINALS | EXERCISE 22: GRAM POSITIVE NON SPORE-FORMING BACILLI

● Basis whether a person may receive vaccination

against diphtheria or not.
● Individuals susceptible to diphtheria→
vaccinated
● If they are hypersensitive→ vaccinated with
caution
○ Doses of the vaccine shall be reduced
with an increased number of injections.
○ Performed first before antitoxin therapy
since antitoxin is normally of horse
origin, which may cause hypersensitivity

POSITIVE RESULT:
PROCEDURE
● White precipitin lines extending from the line of
bacterial growth, forming an angle of about 45
degrees C→ Toxigenic ● Involves injection of:
○ 0.1 mL of diphtheria toxin on the Test Arm (TA)
○ 0.1 mL of diphtheria toxoid on the Control Arm
(TA)
■ Toxoid- Vaccine preparation or
heat-inactivated toxin whose toxicity
is destroyed but retains capacity to
elicit allergic reactions
● Injection site (TA & CA)
○ Inspected daily up to the 6th day for
erythema, induration or necrosis which
consists of a positive reaction

INTERPRETATION

Positive Reaction
● TA- reaction persists until the 6th day
● CA- no reaction
*Susceptible; Not Hypersensitive

Negative Reaction
IN-VIVO TOXIGENICITY TEST ● TA- no reaction
● CA- no reaction
*Immuned; Not hypersensitive
IN-VIVO TOXIGENICITY TEST
● From a pure culture of an isolate: Combined Reaction
○ Inoculate a 10-ml tube of BHIB ● TA- reaction persists until the 6th day
○ Incubate at 37 degrees C for 48 hours ● CA- reaction peaks at about 48 hours and subsides by
● Control→ Inject one guinea pig with diphtheria day 5
*Susceptible & Hypersensitive
antitoxin
● After 2 hours: Pseudoreaction
○ Inject 5mL of the broth into both the ● TA- reaction subsidies by day 5
control and test animals ● CA- reaction subsidies by day 5
● Observe after 48 hrs *Immuned & Hypersensitive

RESULTS & INTERPRETATION

E. PYRAZINAMIDASE TEST
● C. diphtheria→ test animal dies and the control survives
● Another organism→ both the test and control show PYRAZINAMIDASE (PYZ) TEST
illness or die ● Pyrazinamide is converted into pyrazinoic acid
● Purpose:
○ Helpful to distinguish C. diphtheria
● PYZ negative from other corynebacterium mostly
positive
● C. diphtheriae- posses pyrazinamide
Aw, poor boo-rat
● PYR → ammonia +pyrazinoic acid/OH
○ Ammonium ferrous sulfate sa taas ng
arrow
SCHICK TEST
Positive result
● Other bacteria
SCHICK TEST ● Formation of orange to red color appearing instantly
● Performed to determine: ● Toxin producer
○ Susceptibility→ due to lack of Negative
● C. diphtheria
antitoxins ● Colorless/gray (no change in color)
○ Immunity→ due to presence of ● Non toxigenic
circulating antitoxins
○ Hypersensitivity to diphtheria
toxin or other proteins of the NITRATE REDUCTION & UREASE TEST
diphtheria cell
● Detection of exotoxin NITRATE REDUCTION & UREASE TEST
● Check after 24h up to the 6th day of ● Diptheroids mimic C. diphtheria
injection ○ Hence are differentiated by nitrate
reduction and urease test

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 FINALS | EXERCISE 22: GRAM POSITIVE NON SPORE-FORMING BACILLI

ORGANISM NITRATE UREASE

● Cold enrichment technique:
REDUCTION ○ Recovery of the organism from clinical
samples is enhanced by inoculation in
C. ulcerans - + broth medium and incubation at 4
degrees Celsius for 4 hours prior to
C. pseudodiphthericum + +
incubation on primary culture plates
● Requires 5% to 10% CO2 for growth
C. xerosis + -
● Media:
C. minutissimum - - ○ 5% sheep BA,
○ McBride Agar
C. jeikeium - -

5% SHEEP BAM
LABORATORY DIAGNOSIS: LISTERIA
5% SHEEP BAM
MONOCYTOGENES ● L. monocytogenes has a narrow zone of
beta-hemolysis on sheep BAM
LISTERIA MONOCYTOGENES ● Small transparent and glistening colonies that be
● Gram positive short bacilli/ coccobacilli alpha or gamma hemolytic
● Motile at 25 degrees C, non-motile at 35 degrees
C
○ Tumbling motility observed in wet
mounts
● Can be found singly or in chains
● Ferments Glucose
● VP positive
● Catalase positive
● Esculin positive
● Mistaken as pairs and pneumococci
● Sensitive at gram staining
○ As they can over-decolorize and may
may appear as gram -
● Inoculated in BAM and shows narrow zone of MCBRIDE AGAR
b-hemolysis
MCBRIDE AGAR
● Used as a plating
medium with or
without
supplementation
of blood
● Particle selectivity
is provided by
lithium chloride,
glycine, and
SPECIMENS phenylethanol→
aid in
SPECIMENS suppressing both G+ and G- bacteria other than
● CSF Listeria
● Blood ● L. monocytogenes develops into:
○ Small
● Amniotic fluid/placenta
○ Blue-green colonies
○ With small zone of hemolysis around and
I. MICROSCOPY under colonies

III. MOTILITY TEST

GRAM STAINING
MOTILITY TEST
GRAM STAINING
● Short gram-positive bacilli (coccobacilli) may ● In semi-solid medium:
occur intracellularly or extracellularly ○ Identification can be made
● May occur in pairs and be mistaken for by observing for a thin,
pneumococci umbrella-like growth from
● Tends to appear gram-negative when over the stab line
decolorized. ● Presumptive test for listeria

IN WET MOUNTS
IV. ANTON TEST
WET MOUNTS
● Observed at 25 degrees Celsius ANTON TEST
○ Exhibits “tumbling” or “head
● Pathogenicity test that differentiates pathogenic
over heels” motility
from non-pathogenic strains of L.
monocytogenes.
● Involves instillation of a 24 hr broth culture of L.
monocytogenes in the conjunctiva of a lab
II. CULTURE animal

CULTURE

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 FINALS | EXERCISE 22: GRAM POSITIVE NON SPORE-FORMING BACILLI

● Development of purulent conjunctivitis within 24

to 36 hrs constitutes a positive result

V. CAMP TEST SPECIMEN

CAMP TEST SPECIMEN

● CAMP-Positive Listeria ● Tissue biopsy or aspirates from skin lesions
monocytogenes: I. CULTURE MEDIA
○ Inoculated at right
angles to b-hemolytic
CULTURE MEDIA
S. aureus.
● Blood Agar Medium
○ Note the
○ Small, transparent, glistening colonies
arrow-shaped zone of
○ Alpha or gamma hemolytic
weak enhanced
● Columbia Nalidixic Acid Agar (CNA)
hemolysis indicating a
positive CAMP test ● Chocolate Agar Medium (CAM)
● Human Blood Bilayer Tween (HBT)
● Gelatin Stab Culture
EVALUATE ○ Exhibit test tube brush-like or pipe
cleaner appearance
1. For the isolation of Corynebacterium diphtheria is
culture, specimens are obtained from the ff, EXCEPT:
a. Throat LABORATORY DIAGNOSIS:
b. Nasopharynx LACTOBACILLUS
c. Blood
d. Skin lesions
LACTOBACILLUS
● Gram + bacilli
2. Which of the ff culture media contains coagulated ● Pleomorphic
eggs? ○ Long rods in pairs
a. Columbia CNA
○ Coccobacilli
b. Tinsdale agar
○ Spiral forms
c. Cystine-tellurite blood agar
d. Loeffler’s slant ● Non-branching Catalase negative

3. Brown halos around colonies on Tinsdale agar I. CULTIVATION

indicates:
a. Hemolysis of red blood cells CULTIVATION
b. Tellurite reductase activity
● Blood Agar Medium
c. Cystinase activity
d. Toxin production ● CAM
● CNA
4. Elek test is performed on a Corynebacterium ● Incubated at 35 degrees C, 5-10% CO2 within 48
diphtheria isolate to detect: hours
a. Diphtheria toxin expression
b. Four distinct colony types
c. Weakly B-hemolytic reaction of biotype mitis II. BIOCHEMICAL TESTING
d. NOTA
CATALASE NEGATIVE
5. Which test/characteristic can be used to distinguish ● Differentiate Lactobacillus from Viridans
between Corynebacterium ulcerans and Corynebacterium streptococci
pseudotuberculosis?
a. Presence of polyphosphate inclusions
b. Black colonies on CTBA LACTOBACILLUS VIRIDANS
c. Urease test STREPTOCOCCI
d. Production of acid from starch or glycogen
Growth in Chains of Rods Chains of cocci
Thioglycollate Broth
LABORATORY DIAGNOSIS:
ERYSIPELOTHRIX RHUSIOPATHIAE 10u Penicillin disk
- Perform gram
Long bacilli Coccoid Forms

stain on
ERYSIPELOTHRIX RHUSIOPATHIAE colonies
● Gram + bacilli outside ZOI
● Pleomorphic
● Can form short or long filaments
● Non-branching catalase negative LABORATORY DIAGNOSIS: GARDNERELLA
● Oxidase negative VAGINALIS
● Decolorizes easily → may appear gram variable
GARDNERELLA VAGINALIS
● Gram variable pleomorphic

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 FINALS | EXERCISE 22: GRAM POSITIVE NON SPORE-FORMING BACILLI

● Clue cells:
○ Large squamous epithelial cells
○ Numerous attached small rods
● Non-branching
● Catalase negative
● Oxidase negative

I. CULTIVATION

CULTIVATION
● SPS-free medium/SPS medium w/ gelatin
○ BAM
○ CAM
○ CAN
● Direct isolation from female genital tract
○ HBT
■ B-hemolytic colonies
■ Contains human blood
■ Used for recovery of this
organism
● Incubation:
○ Incubated at 35 degrees Celsius, 5-10%
CO2 within 48 hours

BACTE | ASHERAH D. 7