Notre Dame of Dadiangas University PCMT 30: CLINICAL BACTERIOLOGY

First Semester SY ‘24-‘25 Mother Mary Grace J. Lapiad, RMT, MSMT, CBO, SO1

BIOCHEMICAL IDENTIFICATION OF BACTERIA TRIPLE SUGAR IRON AGAR

➢ Triple Sugar Iron agar (TSI) or Kligler Iron Agar (KIA) TSI and Kligler Iron Agar - useful in identification of gram-negative
- Determine glucose and lactose, or sucrose, utilization enteric bacteria, particularly screening for intestinal pathogens.
(sucrose in TSI only) and hydrogen sulfide production. TSI contains sucrose in addition to lactose and glucose
➢ Methyl Red and Voges-Proskauer tests
- Determine end products of glucose fermentation • Ferrous sulfate and Sodium thiosulfate - detects production
➢ Indole Test of hydrogen sulfide.
- Determine if indole if formed from tryptophan by • Phenol red - used as pH indicator.
tryptophanase • TSI - detects the ability of microorganism to ferment glucose,
➢ Urease test lactose and sucrose.
- Determine hydrolysis of urea • KIA - detects the ability of microorganism to ferment
➢ Simmons Citrate carbohydrates, glucose and lactose.
- Determine if citrate can be used as the sole carbon
source REACTION ON TSI AGAR OR KIA
➢ Carbohydrate Fermentation
1. No Fermentation
➢ Lysine Iron Agar (LIA)
- (ALK/ALK or K/K); (ALK/no change or K/NC)
- Determine if lysine decarboxylase activity
- not members of the family of Enterobacteriaciae.
➢ Sulfide-indole-motility (SIM) or Motility-indole-ornithine
- Organism can degrade peptones present, results in the
(MIO) Media
production of alkaline byproducts in slant or deep
CARBOHYDRATE UTILIZATION - changes indicator to a deep red color
2. Glucose fermenters only, no lactose (sucrose in TSI)
Two enzymes necessary for bacterium to take up lactose and cleave fermentation
into monosaccharides: - K/A
- change indicator to yellow
• Beta-galactosidase permease
- reading results after fewer than 12 hrs of incubation
- serves as a transport enzyme that facilitates entry of
gives false appearance of an organism.
lactose molecule across the bacterial plasma
3. Lactose (and/or sucrose) fermentation
membrane.
- A/A
• Beta-galactosidase
- Glucose fermenters will attack simple sugar glucose
- enzyme that hydrolyzes lactose into glucose and
first and then lactose.
galactose
4. Hydrogen Sulfide Production
• Late or delayed lactose fermenters
- K/A, H2S or A/A, H2S
- bacterial species that lacks beta-galactosidase
- H2S production is a two-step process
permease but possess beta-galactosidase.
- First step, H2S is formed from sodium thiosulfate
• Facultative Anaerobes - Second step, ferrous sulfate visually detects its
- bacteria that can grow either aerobically or
production.
anaerobically
5. Gas Production (Aerogenic) or No Gas Production
OXIDATION-FERMENTATION TESTS (Nonaerogenic)
- results in the formation of bubbles or splitting of the
Differentiate members of Enterobacteriaciae, glucose fermenters medium in the butt or complete displacement of the
from the aerobic pseudomonads and similar gram-negative bacteria. medium.

• Carbohydrate fermentation test

- determine the ability of microorganism to ferment a
specific carbohydrate incorporated in basal medium.
• End product: acid or acid w/ gas.
• Mixed acid fermenters
- bacteria that produces several different acid (lactic
acid, propionic acid, succinic acid)

Dale Ceriales, LMSC ✰

Summary: d. K/K
• NLF, NSF, NGF, No Gas No H2S
• Pseudomonas aeruginosa, Alkaligenes

2. LIA (Lysine Iron Agar)

o Contents: Small amount of protein
o Amino Acid: LYSINE
o CHO: Glucose
o pH Indicator: Bromcresol Purple
o H2S Indicator: Ferric Ammonium Citrate

➢ SLANT: Lysine Deamination

(+) = RED (R)
(-) = Purple (K)
Organisms: PPM
• Proteus
• Providencia
• Morganella

BIOCHEMICAL TEST
➢ BUTT: Lysine Decarboxylation
1. TSI (Triple Sugar Iron) Agar
SAMPLE REACTION
o Contents: Protein source
o CHO:
a. K/K H2S +
• lactose = 10%
(-) Lysine Deam
• Sucrose = 10%
(+) Lysine Decarb
• Glucose = 1%
(+) H2S SALMONELLA
o pH Indicator = Phenol Red
o H2S Indicator = Ferrous Sulfate b. K/A – SHIGELLA
c. R/A – PPM
➢ SLANT: Lactose and/or Sucrose Fermenters
Positive = Yellow (Acid) Ortho-Nitrophenyl-B-D-Galactopyranosidase Test
Negative = Red (Alkaline
➢ BUTT: Glucose Fermenter • ONPG and PNPG test
Positive = Yellow - determine whether the organism is a delayed lactose
Negative = Red fermenter or a true nonlactose fermenter.
➢ Gas Producer = CRACKS • B-galactosidase hydrolyzes ONPG, a colorless compound, into
- galactose and o-nitrophenol, a yellow compound.
- All members of Enterobacteriaceae are glucose • ONPG remains colorless if organism is an NLF.
fermenters and aerogenic EXCEPT Shigella
➢ H2S Producer = Blackening of Medium (SPACEd)

SAMPLE REACTION

a. A/Ag H2S – or A/A

• LF, SF, GF, (+) Gas, (-) H2S
• EKE (E. coli, Klebsiella, Enterobacter)

b. K/Ag H2S + or K/A

• NLF, NSF, GF (+) Gas, (+) H2S METHYL RED TEST
• Salmonella, Proteus
• The broth should be incubated 3 to 5 days.
c. K/A H2S – or K/A • If glucose is metabolized by mixed acid fermentation pathway,
• NLF, NSF, GF, Gas (-), H2S (-) stable acid end product is produced w/c results in low pH.
• Shigella • A red color develops after addition of methyl red indicator.

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 CITRATE UTILIZATION

• Citrate Test - determines whether an organism can use sodium

citrate as a sole carbon source.
• Simmons Citrate Medium - frequently used to determine
citrate utilization.
• INDICATOR: Bromthymol BLUE.
• Ammonium salts - sole nitrogen source.
• Christensen Citrate Medium - alternative test medium
- This medium incorporates phenol red (pH indicator) &
organic nitrogen.
VOGES-PROSKAUER TEST - Alkaline pH, indicator turns from yellow to pink.

• After incubation, a-naphthol is added as a catalyst or color

intensifier.
• 40% KOH and NaOH is added and gently shaken to increase
oxygenation.
• Acetoin is oxidized to diacetyl in w/c in the presence of a-
naphthol and KOH forms red complex.

DNAse

• DNA is a polynucleotide composed of repeating purine and

pyrimidine mononucleotide monomeric units.
• DNAse test medium usually contains 0.2% DNA
• A heavy inoculum of bacteria is streaked onto the surface of
medium in a straight line.
• The plate is incubated at 35 C for 18 to 24 hours.
DECARBOXYLASE AND DIHYDROLASE TESTS • Oligonucleotides formed from the action of DNAs will dissolve
in the acid, forming a clear zone (halo) around inoculum.
• Decarboxylase Test - determine whether bacterial species
possess enzymes capable of decarboxylating (removing GELATIN LIQUEFACTION
carboxyl group)
• Ornithine and Lysine - two amino acid commonly used. • Gelatin - a protein derived from animal collagen
• Moeller decarboxylase base medium – a test to detect • Gelatinase - proteolytic enzymes that break down gelatin into
decarboxylation. amino acids.
- Contains glucose, peptone • Its activity is detected by loss of gelling (liquefaction) of gelatin.
- Two pH indicators: Bromcresol purple and cresol red • Gelatinase activity is affected by size of inoculum and
incubation temperature.
DEAMINASE TEST
INDOLE PRODUCTION
• Phenyalanine deaminase test - determines whether an
organism possess the enzyme that deaminates phenylalanine • Indole - is one of the degradation products of amino acid
to phenylpyruvic acid. tryptophan.
- Helpful in initial differentiation of Proteus, Morganella, • Ehrlich Indole Test - indole is extracted from the broth culture
and Providencia organisms w/c are positive. by addition of 1 ml xylene.
• Kovac reagent - contains PDAB, it does not use a xylene
extraction.
• Ehrlich method - more sensitive than Kovac reagent.

Dale Ceriales, LMSC ✰

 LYSINE IRON AGAR SLANT

• LIA - is a tubed agar slant

- contains amino acid lysine, glucose, ferric ammonia citrate
and sodium thiosulfate.
- Determine whether the bacteria decarboxylate or
deaminate lysine.
- pH indicator: Bromcresol purple

MALONATE UTILIZATION

• Malonate Test - determines whether the organism is capable

of using sodium malonate as its sole carbon source.
• pH indicator: Bromthymol blue

NITRATE AND NITRITE REDUCTION

• Nitrate Reduction Test - determines whether the organism has

the ability to reduce nitrate to nitrite and further reduce nitrite
to nitrogen gas.
• Red color indicates presence of nitrite.
BIOCHEM TESTS

OXIDASE

• Oxidase Test - determines the presence of the cytochrome

oxidase system that oxidizes reduced cytochrome w/
molecular oxygen.
• Helpful in differentiating between the Enterobacteriaceae,
oxidase negative and pseudomonads w/c are oxidase positive.
• Modified Oxidase test - used to distinguish Staphylococcus
from Micrococcus.
• Lavender color w/in 10 to 15 sec is a positive reaction. MOTILITY-INDOLE-ORNITHINE AGAR

UREASE • MIO Agar - is a semisolid agar medium used to detect motility

and indole and ornithine decarboxylase production.
• Urease Test - determines whether a microorganism can - useful in differentiating Klebsiella spp. from
hydrolyze urea, releasing a sufficient amount of ammonia to Enterobacter and Serratia.
produce a color change by a pH indicator. • Ornithine decarboxylation is indicated by a purple color
• Christensen Urea Agar - generally preferred. throughout the medium.
- pH indicator: Phenol Red • Indole production is detected by the addition of Kovac
- the resulting alkaline pH from hydrolysis of urea is reagent; pink to red color is formed in the reagent area if
indicated by a bright pink color. positive.

SULFIDE-INDOLE-MOTILITY AGAR

• SIM Medium - is a semisolid agar helpful in differentiating

gram-negative bacteria in the family Enterobacteriaciae.
• Cloudiness spreading from the inoculation line is positive for
motility.
• The production of H2S indicated by black precipitate and pink
to red after addition of Kovac reagent is positive for indole.

Dale Ceriales, LMSC ✰

 Notre Dame of Dadiangas University PCMT 30: CLINICAL BACTERIOLOGY
First Semester SY ‘24-‘25 Mother Mary Grace J. Lapiad, RMT, MSMT, CBO, SO1

NEISSERIA  Perihepatitis (Fitz-Hugh-Curtis syndrome) - Inflammation of the

GENERAL CHARACTERISTICS membrane lining the stomach (peritoneum) and the tissues surrounding
➢ Aerobic, non-motile, non-spore forming, gram negative diplococci, the liver (perihepatitis)
except: (WEB)
N. weaveri,
 OPTHALMIA NEONATORUM
N. elongata
o Gonococcal eye infection
N. bacilliformis
o Acquired during delivery through an infected birth canal
➢ kidney or coffee-bean shaped diplococci
o Can lead to blindness
All are catalase positive and cytochrome oxidase positive, except:
o Treated with:
N.elongata, & N. bacilliformis (EB)
✓ Crede’s prophylaxis – 2% silver nitrate
➢ Capnophilic and humidophiles (increased moisture)
✓ Erythromycin drops
➢ Natural habitat: Mucus membranes of the RT and urogenital tracts
➢ Primary pathogens: N.gonorrhoea & N.meningitidis
➢ Blood dissemination – less than1%
➢ Fastidious: Require iron for growth
o Purulent arthritis, Septicemia, Fever and rash on the extremities
o Mostly caused by AHU strain
 N. gonorrheae: requires cysteine, X (hemin) & V (NAD) factors:
➢ In blood culture: Inhibited by SPS in blood culture
✓ X factor – derived from: LYSED RBCs
➢ Gelatin added to media to neutralize SPS
✓ V factor – can be derived from RBCS but heat labile. Abundantly
produced by some bacteria (Staphylococcus aureus) and can be
LABORATORY DIAGNOSIS
obtained from yeast, yeast extracts and potato extracts.
➢ Sources of Specimens: genital sources (urethra in men & endocervix
in women) or from other sites (rectum, pharynx, joint fluid)
VIRULENCE FACTORS
➢ In men: when no apparent discharge, swab is inserted up to 2cm in the
➢ Receptors for human transferrin
anterior urethra and slowly rotated to collect
➢ Capsule (N. meningitidis only)
➢ Pili (fimbriae)
 RECTAL SWAB
➢ Cell membrane proteins:
• Swabs for RECTAL CULTURE should be inserted 4-5 cm into the
1. Por (protein I) - channel for passage of nutrients and waste
anal canal
products
o Calcium alginate & cotton swabs – Inhibits N. gonorrheae –
✓ Coded by PorA and PorB genes
DACRON swabs are preferred
✓ PorB expressed only by N.gonorrheae – protective against
o Must be plated immediately to selective media due to extreme
host’s inflammatory response and complement mediated
susceptibility to drying and temperature changes
killing
2. Opa (protein II) – facilitates adherence of organisms to phagocytic
and epithelial cell.  COMMERCIAL TRANSPORT SYSTEMS
3. Reduction modified protein (protein III) – blocks host IgG • Contain Selective Media and CO2 atmosphere
➢ LOS (Lipooligosaccharide) – contains the endotoxin o JEMBEC plates - James E. Martin Biological Environmental
➢ IgA protease – hydrolyses sIgA in mucus membrane Chamber
o Gono-Pak
Neisseria gonorrheae o Transgrow
DISEASE ASSOCIATION  DIRECT MICROSCOPY EXAMINATION
➢ Causative agent of gonorrhoea – acute pyogenic infection of the non- o Smears are prepared from UROGENITAL SPECIMENS •
ciliated columnar and transitional epithelium. Nasopharyngeal specimens – not recommended from gram stain
o Flow of seed due to the presence of commensal Neisseria
o Confused with syphilis o Significant finding: PRESENCE OF GRAM NEGATIVE,
o Also known as the “clap” INTRACELLULAR DIPLOCOCCI
➢ Primarily acquired by sexual contact and occur primarily in the urethra, o Avirulent forms - extracellular
endocervix, anal canal, pharynx & conjunctiva.
➢ May cause DGI – disseminated gonococcal infection
➢ Incubation period of 2-7 days
➢ In men: Purulent discharge and dysuria
o 3-5% of cases are asymptomatic
o Due to: AHU strain (requires: Arginine, Hypoxanthine, and
Uracil)
➢ In women: common site is the endocervix – cervical discharge,
dysuria, & lower abdominal pain
➢ 50% of women are asymptomatic
➢ Complicates to: PIV (pelvic inflammatory disease)
o Sterility
o Ectopic pregnancy

Dale Ceriales, LMSC ✰

 CULTURE AND INCUBATION o 1% of individual CHO
o Does not grow well on BAP ✓ Glucose
o Medium of choice: ENRICHED CAP ✓ Maltose
o Incubation: 35C in a 3-5% CO2 ✓ Sucrose
o Provision of sufficient humidity ✓ Lactose
o Moisture evaporating from the media in a closed jar
o CO2 incubator with humidifier o Result: Yellow color in the medium as a result of fermentation
o Pan of water place at the bottom of the incubator\ ❖ N. gonorrheae: ferments GLUCOSE
o Examined for growth for 7 days
OTHER TESTS
 Chromogenic Substrates Methods of CHO utilization
o Based on colour changes after bacterial enzymes hydrolyse the
CHO substrates
o Useful for identification of N. gonorrheae with aberrant CHO
utilization CTA Test

 Multitest Method
o Multitest conventional chromogenic enzyme method
o Combine substrates of other biochemical tests
o Can also identify other genera such as Haemophilus spp

o Colonies are small, gray to tan, translucent, and raised after 24-48  Matrix-Assited Laser Desorption/Ionization – Time-of-Flight Mass
hours of incubation Spectrometry (MALDI-TOF MS)
o 5 colony types: o Emerging Technology
a) T1 and T2: piliated, virulent colonies o Microbial proteins are separated based on size and charge
- Smaller and raised, appear bright in reflected light o Detects unique protein signatures of the organisms
b) T3-T5: non piliated, a virulent
- Larger, flatter colonies  Non culture Methods
c) AHU colonies: smaller colonies and grow slowly o Detects gonococcal antigens or nucleic acids directly from
o Fresh culture is recommended for workup since the organism can specimen
produce their own autolytic enzyme.
 MICROSCOPIC EXAMINATION FOR ISOLATES ANTIMICROBIAL RESISTANT
o Gram stain of colonies obtained from selective media o Previously susceptible to Penicillin
o Observe for: gram negative diplococci (no PMNs since it’s the o Penicillinase-Producing Neisseria gonorrhoea (PPNG) - resistant to
colonies that are stained!) penicillin
o Plasmid mediated penicillin resistance
PRESUMPTIVE BIOCHEMICAL TEST o Plasmid mediated and chromosomally mediated resistance to:
 OXIDASE TEST – done on all suspected isolates of Neisseria spp. tetracycline, spectinomycin, and fluoroquinolones
o CEFTRIAXONE & CEFIXIME – CURRENT RECOMMENDED TREATMENT

Neisseria meningitidis
GENERAL CHARACTERISTICS
Oxidase Reagent/s: o Can be a commensal in the upper tract!
✓ 1% dimethyl p-phenylenediamine dihydrochloride o Causative agent of: Meningitis and Meningococcemia
✓ 1% tetramethyl p-phenylenediamine dihydrochloride o Recovered from urogenital and rectal sites due to oral-genital contact
Result: Purple color within 10 seconds o Mode of Transmission: CLOSE CONTACT OF INFECTED PERSON VIA
Interpretation: Presence of Neisseria species and related organisms RESPIRATORY DROPLETS
o RISK FACTORS:
✓ Asplenic individuals
✓ Persons with complement deficiencies
✓ Active & Passive smoking
✓ Crowded living condition

DISEASE ASSOCIATION
o Pathogenic strains: Serogroups A, B, C Y, & W-135
DEFINITIVE TEST o Incubation: 1-10 days
o Adheres and colonizes the nasopharyngeal mucosa and enters the
 Carbohydrate Utilization Test using CTA Medium
blood stream:
o Medium: Cystine Trypticase Agar
✓ MENINGOCOCCEMIA: Purpura (hemorrhaging of blood into the
o pH Indicator: Phenol Red
skin and mucous membranes producing bruises) with petechial
skin rash (pinpoint red spot caused by hemorrhage)

Dale Ceriales, LMSC ✰

 ✓ MENINGITIS: Abrupt onset of frontal head ache, neck stiffness, MORAXELLACEAE
confusion, and photopia ✓ Moraxella
o Fulminant form of meningococcemia ✓ Acinetobacter
✓ DIC ✓ Pyschrobacter
✓ Septic shock Note: Moraxella catarrhalis – only isolated in humans and a commensal of
✓ Waterhouse-Friderichsen Syndrome – haemorrhage in the the upper tract
adrenal
Moraxella catarrhalis
DISEASE ASSOCIATION
o Opportunistic pathogen
o Cause of upper RT infection in healthy adults and children
o May cause lower RT infection on persons with COPD
o 3rd most common cause of acute otitis media and sinusitis in children
o May cause life threatening systemic diseases
o Presence of intracellular, gram negative diplococci in RT specimens:
indicate possible infections caused by this organism
o Positive for beta lactamase!
LABORATORY DIAGNOSIS
o Specimens are collected from: CSF, blood, nasopharyngeal swabs & SPECIMEN COLLECTION
aspirates, joint fluids, sputum & urogenital sites 1. Collected from middle ear effusion
o Inhibited by SPS 2. Nasopharynx sinus aspirates
3. Sputum aspirates
 DIRECT MICROSCOPY 4. Bronchial aspirate
o Gram stained smears from specimens
o Expected findings: intracellular & extracellular gram-negative CULTURAL CHARACTERISTICS
diplococci o Grows on BAP or CAP
o Smooth, opaque, gray to white colonies
 CULTURE & INCUBATION o Colonies described as “hockey puck” – remains intact when pushed
o Cultured on BAP and CAP (especially from specimens containing across the plate with a loop
normal microbiota) o Older colonies – “wagon wheel” appearance
o Incubated in the same atmospheric conditions described for N. o Tolerates lower temperature and grows well at 28C
gonorrhoeae and examined daily for 72 hours o Inhibited by Colistin present in the media (Resistant strains may grow)
o OXIDASE POSITIVE; CATALASE POSITIVE
 IDENTIFICATION o ASACCHAROLYTIC – NEGATIVE RESULTS IN CHO UTILIZATION
o Colonies: Medium sized, gray, & convex, & encapsulated strains
are mucoid
o Microscopy: gram negative diplococci
o Presumptive test: Oxidase (+)
o Definitive test: CHO utilization (positive for GLUCOSE and
MALTOSE)

❖ Note: Neisseria meningitidis (Maltose and Glucose)

ALGORITHM FOR LABORATORY IDENTIFICATION TESTS TO DIFFERENTIATE FROM NIESSERIA

o Grows on NA
o DNase positive
o Fails to utilize CHO in the CTA medium
o Butyrate esterase reaction = INDIGO COLOR

NON-PATHOGENIC NIESSERIA
OTHER DIAGNOSTIC TEST

Dale Ceriales, LMSC ✰