Clinical Microbiology Practical - 1

Clinical Microbiology
Clinical specimen collection and transportation

Specimen collection and handling
• Specimens for bacteriology investigation should be forwarded as soon as
possible to the laboratory in leak-proof, sterile containers.
• Neutral glycerol saline should be added to stool sample if there is any delay
before laboratory examination.
• Complete early morning urine specimen (250 ml), for diagnosis of renal
tuberculosis.
• Plain tube (blood) for serology.
• Blood clot may be cultured by adding a selective culture medium, e.g., for
enteric organisms.
• Blood for blood culture (blood culture bottle, liquid, 5 to 19ml, 50 ml). The
blood is injected by insertion of syringe needle through a hole in the cap and
through the central rubber or plastic liner. Don’t remove the cap. Blood culture
at RT, not more than 12 hrs.
• For serous fluids collection (pleural fluid), universal container is used.
• Sputum collected in wide-mouthed disposable container.

 Swabs:
1. Exudate from throat, nostril, ear, skin, wounds. The swab, relatively
inefficient sampling device, should be well loaded with the exudate.
2. Pernasal swabs: used for diagnosis of whooping cough:, secretions in the
nasopharynx.
3. Post-nasal swab: used to sample nasopharyngeal secretion for the diagnosis
of meningococcal carriage.
4. Laryngeal swab: bronchial secretion, for the diagnosis of TB in patients who
cannot expectorate sputum, larynx (coughing and collects the expelled
secretion).
5. Nasopharyngeal and throat washings may yield much better specimens for
virological investigations.
6. High vaginal and cervical swabs, for the diagnosis of gonorrhea and puerperal
fever, a swab should be taken from the uterine cervix and its lumen, rather
than from the general area of the upper vagina.

Transport media
• Used especially if meningococcus or B. pertussis are present (delicate
pathogens). Such as Stuart's medium used, which preserves the viability of the
pathogens, (non-nutrient medium!). The medium is semi-solid, and the swab is
plunged into the depths of the transport medium, the pathogens may remain
alive for a day or two at ambient temperature.
• Deep semi-solid or broth thioglycolate medium for anaerobes.
• Amies transport medium (ATM), semi-solid, contain charcoal, especially for
fastidious organisms (also campylobacter). Without charcoal, for mycoplasma
and Ureaplasma.
• Viral transport medium (VTM).
• Cary & Blair medium, for stool, (salmonella, shigella, vibrio or campylobacter).
• Venkatraman Ramakrishnan (VR): for cholera.
• Sachs buffered glycerol saline: bacillary dysentery.
• Glycerol saline transport medium for typhoid bacilli, faeces specimens.
• Pike's medium, S. pyogenes, pneumococci and H. influenzae.

Specimen rejection criteria
• Unlabeled or incorrectly labeled specimens.
• Specimens received without a request form.
• Request forms received without any specimen.
• Missing vital information in the requisition form.
• Specimens received in improper or non-sterile containers or leaking containers or transport
media.
• Specimen for anaerobes not received in appropriate container.
• Specimen received in fixative (formalin); exception, stool for parasites and ova.
• Specimens received that have been delayed in transit exceeds 2 hours, not preserved.
• The specimen has been transported at the improper temperature.
• Dry swab.
• Unpreserved urine held in the refrigerator for >24 hours.
• Sputum specimen with <25 WBC, >10 epithelial cells/lpf.
• 24-h collection of urine or Sputum for AFB or fungus culture.
• Specimen received for anaerobic culture from a site known to have anaerobes as part of the
normal flora such as mouth, vagina, fistula or intestinal contents, samples from ileostomy or
colostomy.
• Inadequate specimens.

• Glass vessels stoppered with cotton-wool, test tubes stoppered with cotton-
wool or screw-capped bottles can be used.
• Types of media: liquid and solid and semi-solid.
• Disadvantages of liquid media vs solid media: appearance of colonies, useful in
identification, and isolation of pure culture.
• Agar is most commonly to make the solid media.
• pH of media, high if temp falls.
• Gelatin and agar media are melted and distributed while hot, for safety, cool
melted agar media to 55C before distribution. The medium is then sterilized by
heat. (melts at 85 C and solidifies at 32–40 C)
• Liquid medium or molten medium may be distributed in test tubes or bottles.
The test tube is half-filled. The tubes are sterilized by autoclaving.

Sterilization of prepared media
• Autoclave used for thermostable ingredients only.
• Filtration, through a bacterial filter is used for media that contain heat
sensitive ingredients.
• Pouring plates: petri dishes, in sterile precautions.
• Media pH 7.2

Routine laboratory media
1. Basic media or ordinary media: nutrient broth and peptone water, nutrient agar.
2. Enriched media: blood agar (10%), serum agar (Loeffler medium), egg containing media.
3. Enrichment Media (Selective media): neomycin or gentamycin blood agar, selenite F broth
(enteric bacteria), alkaline peptone water for Vibrio spp. For mixed inocula.
• Enrichment media are liquid media containing chemical constituents which inhibit some
normal flora and allow pathogens which may be present in very small number in the specimen
to grow. Isolated colonies of these organisms may be obtained by subculturing onto solid
media.
• In addition, it may differentiate the pathogen from commensals that grow by the color and
opacity of the colonies, e.g. blood tellurite medium for C. diphtheriae.
• Transport media are also frequently used to sustain the viability of organisms when a clinical
specimen is to be transported from the periphery to laboratory. The transport medium
prevents the outgrowth of contaminants during transit and sustains the pathogen. Cary and
Blair and Stuart media are two examples of this group of media.
4. Indicator or differential media: MacConkey agar, urea broth (Metabolic activities).
• Differential media have got some chemical constituents which characterize different bacteria
by their special colonial appearances in the culture, e.g. MacConkey agar contains lactose as a
substrate and neutral red as an indicator. Bacteria fermenting lactose produce acid and this
will change the colour of the indicator and thus the colonies will turn red. The red lactose
fermenting colonies can be differentiated from the pale non-lactose fermenting colonies.

Ingredients of culture media:
• Water, distilled or demineralized water.
• Agar, prepared from seaweeds, as powder. A concentration of 1-2%, dissolved
in liquid medium at 100C for 1 hour or more, most agar melt at 95C and solidify
at 42C.
• Semi-solid media agar concentration 0.2-0.5%. Motile bacteria spread.
• Peptone, water soluble products, from lean meat or protein material like heart
muscle, casein, fibrin, soya flour by using proteolytic enzymes, pepsin, trypsin
or papain> (peptone, amino acids).
• Meat extract,
• Yeast extract,
• Blood, defibrinated, rabbit, horses, sheep, or man.
• Serum, animal sera,

Ready prepared media
• Nutrient broth, cooked meat broth.
• Nutrient agar.
• Blood agar.
• Chocolate (CHOC) agar (75C).
• MacConkey, a medium without NaCl is chosen as this inhibits the
spreading of proteus spp, MacConkey without salt.
• Sensitivity test agar, MH agar.
• Thioglycolate medium for anaerobic bacteria (except the surface
of it).
• MacConkey contain bile salt to inhibit non-intestinal bacteria,
lactose with neutral red as indicator.

Media for blood culture
• Brain-heart infusion broth with cooked meat
particles (BHI/CMP): for aerobic and anaerobic
bacteria.
• Brain heart infusion broth: aerobic bacteria.
• Thioglycolate broth, support growth of anaerobes
and aerobes.
• Castaneda medium, solid and liquid phases, for
the isolation of brucella and salmonella from
blood.
• Saponin broth, for viridians streptococci.
• Liquoid broth, contain Sodium polyanethole
sulfonate (SPS) anticoagulant.

Blood should be collected in the following clinical conditions:
• Enteric fever
• Subacute bacterial endocarditis
• Brucellosis
• Any Septicemias.
• For enteric fever blood should be collected in the first week of the
illness.
• For septicemias, 4 frequent samples should be collected at
subsequent temperature in the same day.
• The Samples of blood should be sent to the laboratory
immediately.
• Note: blood sample collected prior to antibiotics administration.

Wires used for inoculation
• Original type of inoculating wire was of
platinum.
• The wire is sterilized by Bunsen flame (red-
hot).
• Loop, circular and completely closed loop, it is
the most useful of the inoculating wires (solid
culture and liquid).
• Pre-sterilized disposable loops and other
applicators are made of plastic used where
Bunsen burners cannot be used.
• Straight wire, used for stab cultures and also
for picking off single colonies.

Methods for isolation of a pure culture
• Solid culture.
• Tellurite media for diphtheria.
• Enrichment media: Selenite F broth for salmonella, willis and
Hobbs medium for clostridium spp.
• Sputum for culture of mycobacterium, is treated with a chemical
such as NaOH or saturated trisodium phosphate, to kill
accompanying bacteria. (Decontamination)
• Animal inoculation.

Disposable of culture
• Sterilized, even negative cultures.
• Incineration, autoclaving and chemical disinfection for
sterilization.
• Autoclaving for TB, and sporing bacteria, Clostridium
spp, Bacillus anthracis.
• Incineration for disposable culture containers.

• Ferric ammonium
citrate, H2S.
• Neutral red
red/pink at acidic
pH.

Xylose-lysine-deoxycholate agar
• Phenol red,
yellow at acidic
pH.
• Ferric
ammonium
citrate for H2S.

• Neutral red,
pink at
acidic pH.
• Ferric
ammonium
citrate, H2S.

• Phenol red,
yellow at acidic
pH.

Thiosulfate-citrate-bile salts-sucrose agar
• Bromothymol blue,
yellow at acidic pH.

• Neutral red, pink
at acidic pH.

• Cystine lactose
electrolyte
deficient (CLED).
• Bromothymol
blue: yellow at
acidic pH.

• Bromothymol blue:
yellow colonies LF
at acidic pH.
• Ferric ammonium
citrate: H2S
indicator.

Procedures and biochemical identification of bacteria
Plating Procedures:
• The clinical specimen and the suspected pathogens will determine the
selection of the primary plating media.
Clinical specimens:
1. Blood:
• Blood is normally sterile.
• Bacteremia: Bacteria in the blood
• Septicemia: Bacteria and their toxins increasing in numbers in the blood
causing harm to the patient
• When drawing blood cultures, avoid skin contamination and collect sample,
if possible, before antimicrobial therapy.
• Bacteria are in highest numbers in the blood just before fever spikes. It is
important to collect several specimens at different times for greatest
potential of bacterial yield (sensitivity). The volume of blood collected
probably has the greatest effect on isolation of bacteria (10-30 ml).

• Cultures:
• Blood culture systems utilize bottles containing liquid media.
• Generally, two bottles are inoculated: one for aerobes and one
for obligate anaerobes. However, because of the reported
decrease in the incidence of anaerobic bacteremias, a number of
hospitals have stopped using anaerobic bottles.
• Most aerobic bottles contain 5-10% CO2 .
• Blood culture bottles often contain sodium polyanethol
sulfonate (SPS), an anticoagulant that also inhibits
complement and inactivates neutrophils.

2. Cerebrospinal fluid:
• CSF surrounds the brain and spinal cord and carries nutrients and waste; it is
normally sterile.
• Meningitis is an inflammation of the meninges.
• Encephalitis is an inflammation of the brain.
• The most common isolates found in CSF are Neisseria meningitidis, Streptococcus
pneumoniae, Streptococcus agalactiae, E. coli, Staphylococcus aureus, and Listeria
monocytogenes.
• Diagnoses are made by a direct Gram stain and culturing on SBA, MAC, and
chocolate agars.
• CSF should be collected (lumbar puncture) under sterile precautions 2-5 ml of it can
be sent to the laboratory in sterile test tubes or screw capped bottle without any
delay!
3. Throat:
• S. pyogenes (group A Streptococcus) is the most important pathogen isolated in
throat cultures; group B streptococci.
• Alpha-hemolytic streptococci viridans group, Neisseria spp., Corynebacterium spp.,
and coagulase negative staphylococci make up the majority of the normal oral flora.
• Culture on SBA and other media as needed by special request.
• Throat Swab:
• Two swabs to the laboratory, one for smear and one for culture.
• Swab the back of the throat; without touching the other parts of the mouth.

4. Nasal, Nasopharyngeal and Oral Swabs:
• Nasal, nasopharyngeal and oral swabs should be collected in the same way as
throat swabs.
5. Ear Swab
• Two swabs should be taken, one for the mycological examination another for the
bacterial cultures.
6. Sputum:
• Used to diagnose lower respiratory tract infections (e.g., pneumonia).
• The lower respiratory tract is normally sterile. However, sputum from the lungs
acquires normal flora passing through the oral cavity.
• A direct Gram stain is performed to determine the quality of the specimen.
Acceptable specimens are cultured on SBA, MAC, and chocolate agars.
• Typically, squamous epithelial cells are an indication of contamination with oral
flora, whereas polymorphonuclear cells (PMNs) indicate a quality specimen. A
general rule for an acceptable specimen might be <10 squamous epithelial cells
and >25 PMNs/low power field. This does not pertain to neutropenic or atypical
pneumonia samples, which often have non-purulent sputum.
• The sputum should be collected in sterile screw container.

Common significant sputum isolates:
1. Streptococcus pneumoniae is an important cause of community acquired
pneumonia, and it is the most common cause of pneumonia in geriatric
(elderly) patients.
2. Klebsiella pneumoniae is associated with nosocomial pneumonia and
pneumonia in alcoholics.
3. Staphylococcus aureus causes community-acquired and nosocomial
pneumonia, usually secondary to another infection or predisposing factor.
4. Pseudomonas aeruginosa causes nosocomial and severe pneumonia in
patients with CF.
5. Haemophilus influenzae causes infection in infants, children, and the
immunosuppressed. The incidence of infections has decreased since
routine use of the Hib vaccine.
6. Legionella pneumophila primarily infects middle-aged males. Legionella
spp. will not grow on routinely used media (i.e., SBA, chocolate, and
MAC). Grow in BCYE.
7. Mycoplasma pneumoniae causes primary atypical pneumonia, which is
mostly seen in young adults. Mycoplasma will not grow on routinely used
media.

7. Urine:
• Urine is normally sterile.
• Bacteriuria is bacteria in the urine.
• Calibrated loops (0.01 ml or 0.001) are used to determine colony counts on media.
• Urine specimens are generally plated onto SBA and MAC or EMB.
• Common significant urine isolates include E. coli, Klebsiella spp., Enterobacter spp.,
Proteus spp., Staphylococcus aureus, Staphylococcus saprophyticus, Enterococcus
spp., Pseudomonas aeruginosa, and yeast.
• In females: Midstream urine sample should be collected after cleaning the external
genitalia with soap and water. On some occasions catheterization should be done, but
always avoid catheterization since there is a chance of introducing microorganisms into
the bladder.
• In males: The glans is cleansed with soap and water and midstream urine should be
collected.
• Urine specimens should be brought to the laboratory immediately within one hour. If
that is not possible, they should be kept in the refrigerator for not more than 24 hours
time.

8. Stool:
• Feces contain many species of anaerobic and facultative anaerobic normal
flora.
• Bacteria causing gastroenteritis include Shigella spp., Salmonella,
Campylobacter jejuni, E. coli (e.g., 0157:H7), Yersinia enterocolitica,
Clostridium difficile (must test for cytotoxin, culture), and Vibrio spp.
• Selective and differential media are used for the isolation and screening of
specific pathogens.
9. Rectal Swab:
• This can be taken by introducing the sterile swab into the anus, and the
swab should be replaced in a test tube.
10. Genital tract:
• Laboratorians commonly look for Neisseria gonorrhoeae and Chlamydia
trachomatis.
• The cervix is typically a sterile site. The vagina contains normal flora that
changes with age. Lactobacillus spp. are the predominant flora during
childbearing years. Earlier and late in life, staphylococci and corynebacteria
predominate.

Types of genital tract infections:
1. Cervicitis and urethritis usually caused by N. gonorrhoeae and C. trachomatis
2. BV, is due to overgrowth of some species of normal vaginal flora, most likely
Mobiluncus. There is a corresponding decrease in lactobacilli. Gardnerella vaginalis is
considered normal vaginal flora and may only be an indicator of BV.
3. PID is a complication of infection caused by N. gonorrhoeae or C. trachomatis
involving the endometrium or fallopian tubes.
4. Prostatitis is usually caused by enterics.
• Vaginal swab.
• Cervical swab: speculum should be used to visualize the cervix and cervical swab
should be taken under sterile precautions and sent to the laboratory.
• Urethral Swab and Prostatic Fluid: Two samples should be sent to the laboratory,
one for direct smear examination one for culture.
• N. gonorrhoeae need specific selective media (e.g., modified Thayer-Martin).
• Molecular techniques are commonly used for detecting both N. gonorrhoeae and C.
trachomatis.

11. Wounds/abscesses:
1. Superficial skin infections: Staphylococcus aureus and Streptococcus
pyogenes.
2. Folliculitis (hair follicle infection): S. aureus and Pseudomonas aeruginosa.
3. Boils, bedsores, etc.: S. aureus.
4. Impetigo: S. pyogenes and S. aureus.
5. Erysipelis: S. pyogenes.
6. Deep and surgical wounds and abscesses: Anaerobes from normal body
sites.
• Pus from close abscess can be collected by sterile syringe and needle, by
aspiration, afterwards sterile swab is used.
12. Conjunctival Swab:
• The swab should be taken before starting on any local antibiotic therapy; if
started it should be washed with sterile saline and then the swab should be
taken from the suspected area.

• SPECIMENS FOR ACID FAST BACILLI:
• Sputum, urine (24 hours), laryngeal swab and gastric
lavage, pus from abscess, pleural or peritoneal; fluids, CSF,
feces, tissue biopsy.
• SPECIMENS FOR ANAEROBIC CULTURE:
• Blood, pus, urine, serous fluids (pleural fluid).
• Diseases of central nervous system:
• Feces, CSF, throat swabs, neuropsy specimen, blood, saliva.
• Disease of the Eye:
• Sterile swabs.
• Diseases of the Respiratory Tract:
• Throat swabs and nasopharyngeal swabs.

ANAEROBIC BACTERIA
General Characteristics:
• Anaerobic bacteria (i.e., obligate anaerobes) comprise most normal flora of the mucous membranes.
• Suspect anaerobic bacteria in the following situations:
• Foul odor (from gas production) and necrotic tissue.
• Anaerobic body sites, abscesses, and wounds.
• Surgical specimens.
Definitions:
• Obligate anaerobe: Bacterium that cannot use oxygen for metabolism and oxygen is lethal to the
microorganism (Clostridium spp).
• Aerotolerant anaerobe: Bacterium that cannot use oxygen but can grow in its presence.
• Facultative anaerobe These bacteria which can live and grow in the presence as well as in the
absence of oxygen. Most of the bacteria come under this group (Staphylococci, Enterobacteriaceae).
• Obligate aerobe: Bacterium that requires free oxygen for their growth (Moraxella & Brucella).
• Microaerophile: Bacterium that requires oxygen at trace concentrations of 5-10% (Campylobacter, H.
pylori).
• Capnophile: Bacterium that requires increased concentration of CO2 (5-10%) (Neisseria spp, H.
influenzae).
• SOURCE OF ANAEROBES: Anaerobes occur in a variety of human body sites. They are usually present as
normal commensals without causing any harmful effects. During infection, they are isolated from various
specimens like, Faeces, Blood, Pus, Exudates

Anaerobic Media
• Centers for Disease Control and Prevention (CDC) anaerobic blood agar: For general
growth of all anaerobes.
• Bacteroides bile esculin (BBE) agar: Selective and differential medium used for
Bacteroides fragilis
• Kanamycin-vancomycin laked sheep blood (KVLB) agar: Enriched selective medium
for isolation of slowly growing anaerobes such as Bacteroides, laked blood enhances
pigment formation.
• Phenylethyl alcohol (PEA) agar: Enriched and selective medium used to grow most
anaerobes, including Clostridium and Bacteroides; inhibits the growth of facultative
anaerobic, gram-negative bacilli (e.g., Enterobacteriaceae).
• Columbia-colistin-naladixic agar with 5 % sheep blood: Inhibits gram negative
organisms and is used to grow most gram-positive anaerobes and facultative anaerobes.
• Egg yolk agar is used to detect proteolytic enzymes, lipase and lecithinase (Nagler's
reaction) produced by Clostridium. Lecithinase activity produces an opaque zone from
the cleavage of lecithin releasing insoluble fats (diglyceride ). Lipase cleaves lipids,
releasing glycerol, which floats to the top of the medium producing a blue-green sheen
(mother-of-pearl) on the agar surface.

• Broths with reducing agents, such as thioglycolate and cooked (or chopped) meat,
can be used to grow anaerobic bacteria. Sometimes resazurin, an oxidation-
reduction indicator, is added. The indicator is pink in the presence of oxygen and
colorless when reduced.
• Solid media must be placed in anaerobic conditions in order for obligate
anaerobes to grow:
• Commonly used systems include anaerobic GasPak jars and anaerobic hoods. In
the presence of palladium, a catalyst, the following reaction occurs:
• An oxidation-reduction indicator must be used to determine if anaerobic
conditions have been met. Methylene blue is the most commonly used
oxidation-reduction indicator. When anaerobic conditions are achieved, the
methylene blue indicator will turn from blue (oxidized) to white, indicating
reduction. Anaerotest.
• Aerotolerance testing: Before attempting to identify a possible anaerobic
bacterium, it first must be demonstrated to be an obligate anaerobe. A colony is
inoculated to an anaerobic blood agar plate, which is incubated anaerobically, and
to a chocolate agar plate incubated under conditions of increased CO2. Isolates
growing only on the plate incubated anaerobically are obligate anaerobes.

• The candle jar is employed to provide 5-10% CO2 atmosphere for the
cultivation of carboxyphilic bacteria like: Pneumococci, Gonococci, and
Meningococci, etc.

• The bench should be free from dust and wiped with disinfectant at least before the start
and at the end of each day.
• Aerobic culture at 37C, 43C for campylobacter, 30C for Leptospira, 22-28 for many
fungi.
• Culture with added CO2: brucella abortus, capnophilic streptococci, gonococci and
pneumococci (5-10% CO2).
• Culture in a microaerophilic: 5-7% CO2, Campylobacter jejuni, Actinomyces Israelii, H.
pylori.
• Anaerobic culture: obligate anaerobes.
• Anaerobic inoculation: AneroGen, oxygen adsorbing envelope, anaerobic cabinets.

Antimicrobial Susceptibility Testing (AST)
1. Disc method
2. MIC
3. E test.
4. Vitek.
1. Disk diffusion (Kirby-Bauer sensitivity test).
• Mueller-Hinton agar (MHA). In the case of fastidious
microorganisms (e.g., Streptococcus pneumoniae), MHA with 5%
sheep red blood cells is used. For Haemophilus infiuenzae,
Haemophilus test medium (HTM) is used. HTM is Mueller-Hinton
base supplemented with hematin, NAD, and yeast extract.
• Bacterial inoculum, 10(8) colony forming units/mL, which is
equal to a McFarland 0.5 turbidity standard.
• MHA plates are incubated for 18 hours at 35 °C in ambient air.
Both HTM and MHA with sheep red blood cells are incubated in 5-
7% CO2 for 18-20 hours.
• After incubation, the diameters of the zones of inhibition are
measured. The zone sizes are compared to standard interpretation
charts, and the results are reported as sensitive (S),
intermediate (I), or resistant (R).

• Turbidity Standard (1x10(8)-2x10(8) bacterial suspension.
• The turbidity standard solution should be placed in a tube identical to the one
used for the broth sample. It can be stored in the dark at room temperature for
six months, provided it is sealed to prevent evaporation.

• Reading and reporting:
• Measure the inhibition zone, the distance in mm from the
edge of the disc to the zone edge, using calipers, a mm rule
or ruled template.
• Some proteus spp back-swarm into the inhibition zones
during incubation, such growth in the zone should be
disregarded. Swarming can be prevented by adding p-
nitrophenylglycerol.
• Categories of sensitivity:
• Sensitive: the zone size of the test strain is larger
than, equal to or not more than 3 mm smaller than
that of the control strain.
• Resistant: the zone size of the test strain is smaller
than 2 mm.
• Intermediate: the zone size of the test strain is at
least 2 mm, but also 3 mm smaller than that of
control strain.
• Sensitive (S): Infection treatable by the normal dosage of
the antibiotic.
• Intermediate (I): Infection may respond to higher
dosage.
• Resistant (R): Unlikely to respond to usual dosage of the
antibiotics.

2. Dilution tests
• In these assays, bacteria are exposed to different concentrations of antimicrobial agents.
The smallest concentration that inhibits growth of the bacteria is recorded; this value is
the minimal inhibitory concentration (MIC).
A. Broth dilutions: Dilutions of the antimicrobial agents are prepared in broth. The
assays are generally performed in microtiter plates.
B. Agar dilutions: Dilutions of the antimicrobial agents are prepared in agar. Bacteria
are inoculated onto the agar plates.
• The minimum bactericidal concentration (MBC) of an antimicrobial agent is defined as
the lowest concentration of an antimicrobial agent that kills at least 99.9% of the
bacteria in the original inoculum. This can be determined by first performing a broth
dilution test and then subculturing the tubes without visible growth to media without
antimicrobial agents. The sample taken from the tube with the lowest concentration of
antimicrobial agent showing no growth is representative of the MBC.

• Antibiotic disc (stock) store at (-14 to -20C) frozen, working discs at 2-8C for 1 month. M-H
agar (70 days at 4-8C).
• Metronidazole may deteriorate by light! Should be kept in the dark.
• Avoid leaving the inoculated plates at RT before applying discs, as bacteria may multiply, and
this gives smaller zone sizes.
• Avoid leaving plates at RT after discs have been applied, as antibiotic diffusion may result in
larger zone sizes.
• Any visible liquid on agar surface, the plate should invert, ajar on its lid for evaporation.
Alternatively, place the plate in a 35°C incubator or in a laminar flow hood at RT until dry
(10 to 30 minutes).
• Plates should be incubated in air at 35-37C overnight (ideally 16-18 h). Neisseria spp require
incubation in 10% CO2. (Inverted).
• When testing Staphylococcus against oxacillin or vancomycin, or Enterococcus against
vancomycin, incubate for a full 24 hours before reading.
• Recommended temp for methicillin sensitivity test is 30-35C, 48 h incubation (slow growing
of MRSA). This problem can be overcome by incubating the bacteria at 30°C or by using 5%
salt agar and incubating at 37°C.
• In order to ensure that the zone diameters are sufficiently reliable for testing susceptibility to
sulfonamides and co-trimoxazole, the Mueller-Hinton agar must have low concentrations of
the inhibitors thymidine and thymine.

• Each new lot of Mueller-Hinton agar should therefore be tested with a control strain of
Enterococcus faecalis (ATCC 29212 or 33186) and a disc of cotrimoxazole. A satisfactory
lot of medium will give a distinct inhibition zone of 20 mm or more that is essentially
free of hazy growth or fine colonies.
• On removal from the refrigerator, the containers should be left at room temperature for
about one hour to allow the temperature to equilibrate.
• The antibiotic discs may be placed on the inoculated plates using a pair of sterile forceps
or antibiotic disc dispenser (Leave the inoculum to dry for a few minutes at room
temperature).
• Disks should not be placed closer than 24 mm (center to center) on the MH agar plate.
Ordinarily, no more than 12 disks should be placed on a 150-mm plate or more than 5
disks on a 100-mm plate. However, the semiautomatic disk dispensers hold 16 and 8
disks respectively and may not maintain the recommended 24 mm center to center
spacing.
• Each disc should be pressed down gently to ensure even contact with the medium.
• The plates should be placed in an incubator at 35C within 30 minutes of preparation.
Temperatures above 35C invalidate the results for oxacillin/ methicillin.

Control strains
• They should preferably be run every week, or with
every fifth batch of tests, and in addition, every time
that a new batch of Mueller-Hinton agar or a new
batch of discs is used.
• The standard strains are:
• (American Type Culture Collection):
• Staphylococcus aureus (ATCC 25923)
• Escherichia coli (ATCC 25922)
• Pseudomonas aeruginosa (ATCC 27853)
National Collection of Type Cultures

Source of errors
• Too heavy inoculum> smaller zones.
• Too light inoculum> R to S (large zones).
• Expired discs.
• Improper distance b/w discs.
• Too thick agar> small zones.
• Too thin agar> large zones.
• If the pH is <7.2 certain drugs will appear to lose
potency (aminoglycosides, quinolones,
macrolides), while other agents may appear to
have excessive activity (tetracycline). If the pH is
>7.4, the opposite results may occur.
• Excessive thymidine or thymine can reverse the
inhibitory effects of sulfonamides and
trimethoprim resulting in smaller and less
distinct zones of inhibition, or no zones at all.

3. Gradient diffusion: Etest® or
Epsilometer test, MIC test strip:
• Provides quantitative antimicrobial
susceptibility testing results.
• Procedure:
• A bacterial suspension equal to a
McFarland 0.5 turbidity standard
is prepared.
• The bacteria are lawned onto a
Mueller-Hinton agar plate and the
Etest strips (impregnated with
antimicrobials) with MIC scale,
are placed on top of the agar. Each
strip contains a different
antimicrobial agent.
• After incubation, the bacteria
produce an elliptical zone of
inhibition around the strip. The
MIC is read from a scale on the
strip where the zone of inhibition
crosses the strip.

VITEK 2 System. (Courtesy bioMérieux Vitek, Hazelwood,MO.)

Alyazeed Hussein, BSc, SUST
This has been a presentation of Alyazeed Hussein
Thanks for your attention and kind patience
@elyazeed7
@Alyazeed7ussein

Clinical Microbiology Practical - 1

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