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ANTIMICROBIAL SUSCEPTIBILITY TESTING A. Test tube dilution/broth Dilution Method Involves challenging the organism of interest with antimicrobial agents in a broth environment. (Mueller-Hinton Broth) Specific amount of antibiotic is prepared in a decreasing concentration in broth by serial dilution techniques, and standard numbers of the test organism is inoculated. Standard inoculum size: 5 x 10 5 CFU/mL Absence of turbidity of broth signifies inhibition of bacterial growth by the antibiotics being tested. Can be used to determine MIC and MLC concentrations.

Antimicrobial Susceptibility Testing

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Page 1: Antimicrobial Susceptibility Testing

ANTIMICROBIAL SUSCEPTIBILITY TESTING

A. Test tube dilution/broth Dilution Method Involves challenging the organism of interest with

antimicrobial agents in a broth environment. (Mueller-Hinton Broth)

Specific amount of antibiotic is prepared in a decreasing concentration in broth by serial dilution techniques, and standard numbers of the test organism is inoculated.

Standard inoculum size: 5 x 10 5 CFU/mL Absence of turbidity of broth signifies inhibition of

bacterial growth by the antibiotics being tested. Can be used to determine MIC and MLC

concentrations.

Page 2: Antimicrobial Susceptibility Testing

Can be used to determine MIC and MLC concentrations.

A. Microdilution

-broth volume: 0.05-0.1 ml -Schaedler’s broth, West-Wilkins broth, BHI B.Macroddilution -broth volume: 1.0ml or greater

The lowest concentration of the antibiotic without bacterial growth after 16-24 hours of incubation is the minimum inhibitory concentration (MIC)

The lowest concentration of the antibiotic without bacterial growth when subcultured to a fresh medium is the lethal concentration (MLC)

MH broth with 2% NaCl – to improve detection of oxacillin-resistant staphylococci.

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B. Agar Dilution Method The antimicrobial concentrations and organism to be

tested are brought together on an agar-based medium rather than in a broth.

One or more bacterial isolates are tested per plate; MIC can also be determines

Standard inoculation size: 1x10 4 CFU/mL Shelf life of agar dilution is only one week for most

antimicrobial agents. After incubation, the plates are examined for growth. Reference method for testing anaerobes and N.

gonorrheae. Reference method for anaerobes: Brucella agar with laked

blood and vitamin K (Wadsworth Method)

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C. Disk Diffusion Method (Kirby-Bauer Test) Limited to aerobic and facultatively anaerobic

bacteria Filter paper disks impregnated with various

antimicrobial agents of specific concentrations are carefully placed on a agar plate previously inoculated with the bacterium being tested.

Standard inoculum size: 1.5 x 10 8 CFU/mL Susceptibility standard medium: Mueller

Hinton Agar (MHA)

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TURBIDITY STANDARD The use of a standard inoculum size is as

important as culture purity and is accomplished by comparison of the turbidity of the organism suspension with a turbidity standard.

0.5% McFarland Turbidity Standard -9.5ml of 1% H2SO4 + 0.5ml of 1.175% of barium chloride is equivalent to 1.5 x 10 8 colony forming units CFU/ml

Pure cultures are grown or are directly prepared from agar plates to match the turbidity of the 0.5 McFarland standard

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PREPARATION OF PURE CULTURE OF SUSCEPTIBILITY TESTING:

1. Pure inocula (cultures) are obtained by selecting 4-5 colonies of the same morphology, inoculate them into broth medium and incubate for 3-5 hours to achieve a turbid suspension.

2. alternatively, 4-5 colonies 16-24 hours of age may be selected from an agar plate and suspended in broth or 0.85% NSS to achieve a turbid suspension.

3. Matching turbidity using the unaided eye is facilitated by holding the bacterial suspension and McFarland tubes side by side and viewing them against a black-lined background.

4. If the bacterial suspension initially does not match the standard’s turbidity the suspension may be diluted, or supplemented with more organisms, as needed.

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FACTORS AFFECTING ZONE OF INHIBITION1. Thickness of susceptibility agar plate (4-6mm)-if the agar is too thick, zone sizes would be smaller;if agar is too thin, zone sizes would be larger.

2. The amount of inoculum or test organism.

3. The growth rate of the test organism.-air at 35C -37C for most bacteria (16-24 hrs)-5-7% CO2 for N. meningitides-prolonged incubation may result in false resistance interpretation

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4. The pH of the medium (7.2 – 7.4)-decrease pH affects the activity of antimicrobial agents.-aminoglycosides and macrolides may lose potency; penicillin may have increase activity

5. The number of disk per plate.-12 disks per plate-placement of more than 12 disks may result in overlapping zones (erroneous results)

6. The concentration of divalent cations ( calcium and magnesium)-can affect testing of aminoglycosides and tetracyclines against P. aeuruginosa.

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CAUSES OF FALSE RESISTANCE:

1. Use of unduly heavy inocula of cultires or undiluted specimen materials.

2. Late examination of test plates after zones become overgrown.

3. Use of disc with inadequate drug concentration due to prolonged storage, failure to refrigerate as from disc container opened frequently.

4. Use of wrong organism.

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D. Etest Is a dilution test based on the diffusion of a

continuous concentration gradient of an antimicrobial agent from a plastic strip into an agar medium.

The plastic strip is contains dried, predefined concentrations of stabilized drug on one side and a continuous MIC interpretative scale on the other side.

The strip is placed on the surface of the culture medium inoculated with the desired organism.

(+) Result: ellipse of growth inhibition the MIC is read at the point on the scale where the

ellipse intersects the strip. An alternative susceptibility test for fastidious

bacteria (S. pneumoniae and H. influenzae)

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E. SERUM BACTERIAL TEST Determines the activity of one or more

antimicrobial agents present in serum against an organism that has been recovered from the patient.

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METHODS FOR BACTERIAL IDENTIFICATION1. Rapid Methods

A. Manual Biochemical System API 20E

-for the identification of the family Enterobacteriaceae and other gram (-) bacteria.

-it consist of plastic strip with 20 microtubes containing dehydrated biochemical substrates.

-the biochemical subtrates are inoculated with a pure culture suspended in sterile physiologic saline.

-after 5 hours incubation, the 20 test results are converted to a seven or nine-digit profile. 

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B. Mechanized or Semiautomated Automated Systems -Vitek, Microscan Walkaway and Aris C. Immunologic Techniques

Page 14: Antimicrobial Susceptibility Testing

2. Bacteriophage Typing is based on the specificity of pjage surface

receptors for cell surface receptors. only bacteriophages that can attach to surface

receptors can infect bacteria and cause lysis. the bacteria to be tested is inoculated on petri

plate and a drop of suspension from the different phage is used.

lytic bacteriophages cause plaques, which represents infection by the virus, on lawns of sensitive bacteria.

Bacteriophage – is virus that attacks bacterial species

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3. Molecular Methods A. Nucleic Acid-Base Detection Methods - DNA probe technology identifies a microorganism by probing its genetic composition.

- A single-stranded sequence from one organism (probe) is used to search for other microorganism having the same sequence.

- It is utilized for purified DNA preparations, bacterial colonies and specimens such as sputum, tissue, serum and pus.

- Ribotyping – a form of strain typing; ribosomal RNA is used to type bacterial strains by probing chromosomal DNA in Southern blotting

Page 16: Antimicrobial Susceptibility Testing

B. Gas-Liquid Chromatography -it involves analysis of microbial metabolites, cellular fatty

acids, products of pyrolysis of whole bacterial cells.

C. Plasmid Chromatography Identifies microbial isolates of the same or similar strains. Useful for E. coli, Salmonella, Campylobacter and

Pseudomonas strains Considered more accurate as compared to biotyping,

phage typing, antibiotic resistance patterns and serotyping.

Plasmid – is an replicating extrachromosomal molecule of DNA in bacteria

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BIOSAFETY IN A MICROBIOLOGY LABORATORY

Shigellosis, salmonellosis, tuberculosis, brucellosis and hepatitis – are the five most frequently acquired laboratory infections.

Males and younger employees are involved in more laboratory-acquired infections than females and older employees.

The air-handling system of a microbiology laboratory should move air from lower to higher risk areas, never the reverse.

Never recap needles by hand

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DISPOSAL OF HAZARDOUS WASTE All materials contaminated with potentially infectious

agents must be decontaminated before disposal. Infectious waste may be decontaminated by use of an

autoclave, incinerator or alternative waste-treatment methods.

Infectious waste (agar plates, tubes, reagent bottles) should be placed into leak-proof, plastic bags (double bagging)

Pipettes, swabs and other glass objects should be placed into rigid cardboard containers before disposal

Sharp objects (needles and scalpels) are placed in sharp containers which are autoclave or incinerated

 

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BIOLOGICAL SAFETY CABINET

Is a device that encloses a workplace in such a way as to protect workers from aerosol exposure to infectious disease agents.

Air that contains the infectious material is sterilized, either by heat, UV light or by passage through a HEPA filter.

Most hospital clinical microbiology laboratory technologists use class II cabinets

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A. Class I cabinets - Allow room (unsterilized) air to pass into the cabinet and around the area and material within, sterilizing only the air to be exhausted B. Class II cabinets- Sterilize air that flows over the infectious material, as well as air to be exhausted C. Class III cabinets - Provide the most protection to the worker- Air coming into and going out of the cabinet is filter sterilized and the infectious material within is handled with rubber gloves that are attached and sealed to the cabinet. 

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CLASSIFICATION OF BIOLOGIC AGENTS BASED ON HAZARD:

1. Biosafety Level 1 Agents It include those that have no known

potential for infecting healthy people. It is used in laboratory teaching

exercises of students Bacillus subtilis, Mycobacterium

gordonae 

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2. Biosafety Level 2 Agents Are the most commonly being sought

in clinical specimens They include all the common agents of

infectious diseases HIV, Bacillus anthracis, Yersinia pestis

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3. Biosafety Level 3 Agents Mycobaterium tuberculosis, systemic

fungi, Francisella tularensis, Brucella spp.

 4. Biosafety Level 4 Agents Arbovirus, Arenavirus, Filovirus, Small

Pox Virus