Antibiotics

  • Bacitracin
  • Chloramphenicol
  • Erythromycin
  • Kanamycin
  • Nalidixic Acid
  • Nitrofurantoin
  • Novobiocin
  • Optochin
  • Penicillin
  • Streptomycin
  • Sulfamethoxazole
  • Tetracycline

Biochemical tests

  • Acid from glucose
  • Adonitol fermentation test
  • Antibiotic susceptibility tests
  • Arabinose fermentation test
  • Arabitol fermentation test
  • Arginine dihydrolase test
  • Bacitracin susceptibility test
  • Catalase test
  • Cellobiose fermentation test
  • Citrate utilization test
  • Coagulase test
  • Deoxyribonuclease (DNase) test
  • Dulcitol fermentation test
  • Esculin hydrolysis test
  • Fructose fermentation test
  • Galactose fermentation test
  • Gas from glucose
  • Gelatin hydrolysis test
  • Glucose fermentation
  • Glycerol fermentation test
  • Growth in KCN (potassium cyanide)
  • Growth on 6.5% NaCl
  • Growth on 7.5% NaCl
  • Growth on bile salts test
  • Hand Hygiene and Infection Control
  • Hemolysis on blood agar
  • Hydrogen sulfide (H2S) production test
  • Indole production test
  • Inositol fermentation
  • Kirby-Bauer Antibiotic Susceptibility Test
  • Lactose fermentation test
  • Lipase test (using corn oil)
  • Lysine decarboxylase test
  • Malonate utilization test
  • Maltose fermentation test
  • Mannitol fermentation test
  • Mannose fermentation test
  • Melezitose fermentation test
  • Melibiose fermentation test
  • Methyl red test
  • Motility test
  • myo-Inositol fermentation test
  • Nitrate reductase test
  • Novobiocin susceptibility test
  • OF glucose test
  • Optochin susceptibility test
  • Ornithine decarboxylase test
  • Oxidase test
  • Phenylalanine deaminase test
  • Raffinose fermentation test
  • Rhamnose fermentation test
  • Ribose fermentation test
  • Salicin fermentation test
  • Sorbitol fermentation test
  • Starch hydrolysis test
  • Sucrose fermentation test
  • Trehalose fermentation test
  • Urea hydrolysis test (Christiansen’s)
  • Voges-Proskauer test
  • Xylitol fermentation test
  • Xylose fermentation test

Gram negative bacilli

  • Buttiauxella agrestis
  • Cedecea davisae
  • Cedecea lapagei
  • Cedecea neteri
  • Citrobacter amalonaticus
  • Citrobacter amalonaticus biogroup 1
  • Citrobacter freundii
  • Citrobacter koseri
  • Cronobacter sakazakii
  • Edwardsiella hoshinae
  • Edwardsiella ictaluri
  • Edwardsiella tarda (wild type)
  • Edwardsiella tarda biogroup 1
  • Enterobacter aerogenes
  • Enterobacter amnigenus biogroup 1
  • Enterobacter amnigenus biogroup 2
  • Enterobacter cancerogenus
  • Enterobacter cloacae
  • Enterobacter gergoviae
  • Escherichia coli
  • Escherichia fergusonii
  • Escherichia hermannii
  • Escherichia vulneris
  • Ewingella americana
  • Hafnia alvei
  • Klebsiella oxytoca
  • Klebsiella pneumoniae subspecies ozaenae
  • Klebsiella pneumoniae subspecies pneumoniae
  • Klebsiella pneumoniae subspecies rhinoscleromatis
  • Kluyvera ascorbata
  • Kluyvera intermedia
  • Leclercia adecarboxylata
  • Mollerella wisconsinsis
  • Morganella morganii subspecies morganii
  • Morganella morganii subspecies sibonii
  • Proteus mirabilis
  • Proteus myxofaciens
  • Proteus penneri
  • Proteus vulgaris
  • Providencia alcalifaciens
  • Providencia rettgeri
  • Providencia rustigianii
  • Providencia stuartii
  • Rahnella aquatilis
  • Raoultella ornithinolytica
  • Raoultella terrigena
  • Salmonella bongori
  • Salmonella enterica
  • Salmonella enterica subspecies arizonae
  • Salmonella enterica subspecies enterica
  • Salmonella enterica subspecies houtenae
  • Salmonella enterica subspecies paratyphi
  • Salmonella enterica subspecies salamae
  • Salmonella typhi
  • Serratia ficaria
  • Serratia fonticola
  • Serratia marcescens
  • Serratia odorifera biogroup 1
  • Serratia plymuthica
  • Serratia proteamaculans subspecies proteamaculans
  • Serratia rubidaea
  • Shigella dysenteriae
  • Shigella flexneri
  • Shigella sonnei
  • Shimwellia blattae
  • Tatumella ptyseos
  • Yersinia enterocolitica
  • Yersinia frederiksenii
  • Yersinia kristensenii
  • Yersinia pestis
  • Yersinia pseudotuberculosis

Gram positive cocci

  • Dermacoccus nishinomiyaensis
  • Enterococcus avium
  • Enterococcus faecalis
  • Enterococcus faecium
  • Enterococcus gallinarum
  • Kocuria kristinae
  • Kocuria rosea
  • Kocuria varians
  • Kytococcus sedentarius
  • Lactococcus lactis
  • Lactococcus raffinolactis
  • Macrococcus caseolyticus
  • Micrococcus luteus
  • Micrococcus lylae
  • Nesterenkonia halobia
  • Rothia mucilaginosa
  • Staphylococcus aureus
  • Staphylococcus auricularis
  • Staphylococcus capitis
  • Staphylococcus caprae
  • Staphylococcus carnosus
  • Staphylococcus chromogenes
  • Staphylococcus cohnii ssp. cohnii
  • Staphylococcus cohnii ssp. urealyticum
  • Staphylococcus epidermidis
  • Staphylococcus gallinarum
  • Staphylococcus haemolyticus
  • Staphylococcus hominis
  • Staphylococcus hyicus
  • Staphylococcus intermedius
  • Staphylococcus lentus
  • Staphylococcus saccharolyticus
  • Staphylococcus saprophyticus
  • Staphylococcus sciuri
  • Staphylococcus simulans
  • Staphylococcus warneri
  • Staphylococcus xylosus
  • Streptococcus acidominimus
  • Streptococcus agalactiae
  • Streptococcus bovis
  • Streptococcus criceti
  • Streptococcus equi
  • Streptococcus equinus
  • Streptococcus ferus
  • Streptococcus iniae
  • Streptococcus mutans
  • Streptococcus pneumoniae
  • Streptococcus pyogenes
  • Streptococcus ratti
  • Streptococcus salivarius
  • Streptococcus sanguinis
  • Streptococcus sobrinus
  • Streptococcus uberis

Media

  • 6.5% NaCl broth
  • Arginine dihydrolase broth
  • Bile esculin agar slant
  • Blood agar
  • Christiansen’s urea broth
  • DNase agar with methyl green plate
  • Lysine decarboxylase broth
  • Malonate broth
  • Mannitol salt agar
  • MRVP broth
  • Mueller Hinton Agar
  • Nitrate broth with Durham tube
  • Nutrient agar plate
  • Nutrient broth
  • Nutrient gelatin
  • OF glucose broth
  • Ornithine decarboxylase broth
  • Phenol red adonitol broth
  • Phenol red arabinose broth
  • Phenol red arabitol broth
  • Phenol red cellobiose broth
  • Phenol red dulcitol broth
  • Phenol red fructose (levulose) broth
  • Phenol red galactose broth
  • Phenol red glucose (dextrose) broth with Durham tube
  • Phenol red glycerol broth
  • Phenol red inositol broth
  • Phenol red lactose broth
  • Phenol red maltose broth
  • Phenol red mannitol broth
  • Phenol red mannose broth
  • Phenol red melezitose broth
  • Phenol red melibiose broth
  • Phenol red raffinose broth
  • Phenol red rhamnose broth
  • Phenol red ribose broth
  • Phenol red salicin broth
  • Phenol red sorbitol broth
  • Phenol red sucrose (saccharose) broth
  • Phenol red trehalose broth
  • Phenol red xylitol broth
  • Phenol red xylose broth
  • Phenylalanine agar slant
  • Potassium cyanide (KCN) broth
  • Rabbit plasma
  • Simmons’ citrate agar slant
  • Spirit blue agar plate
  • Starch agar
  • Triple sugar iron agar slant
  • Tryptone broth

Reagents, Chemicals, Stains

  • Ammonium Sulfate (saturated)
  • Barritt’s A reagent
  • Barritt’s B reagent
  • Crystal Violet
  • Ferric chloride (10%) reagent
  • Gram’s Decolorizer
  • Gram’s Iodine
  • HCl 0.1N reagent
  • Hydrogen peroxide
  • Iodine reagent
  • Kovac’s reagent
  • Methyl red reagent
  • Nitrate A reagent
  • Nitrate B reagent
  • Oxidase reagent
  • Rabbit Plasma
  • Safranine

Kirby-Bauer Antibiotic Susceptibility Test

The Kirby-Bauer Antibiotic Susceptibility test is used to determine the effectiveness of a collection of antibiotics in preventing bacterial growth through the use of the “disk diffusion method”.  By challenging the growth of the bacteria with a variety of antibiotics, it is possible to determine which antibiotics are effective vs. not effective in preventing growth.  This informs decisions on which antibiotics are of potential benefit for treating a disease caused by this microbe and those that would not be effective.

Plates of Mueller Hinton agar are seeded with cells from a pure culture of bacteria to create a potential lawn.  Next are added in an equidistant pattern paper disks each impregnated with a different antibiotic.  The prepared plates are incubated overnight to allow the bacteria to grow.  During this time, the antibiotics are diffusing away from their paper disks creating a gradient with higher concentrations near the disk and lower concentrations as the distance from the disk increases.  The next day the plates are removed from the incubator and evaluated.  The sizes of the zones of inhibition are measured and compared with standardized charts to determine whether the microbe is resistant, intermediate in resistance, or susceptible to each antibiotic.

The antibiotics commonly used differ for Gram – and Gram + bacteria, in part due to the anatomy and physiology of these two major groups of bacteria.  Often, additional testing using other alternative antibiotics can provide a more refined and focused examination of the resistance and susceptibility patterns for a specific unknown microbe.

Typical Antibiotics Used for Kirby-Bauer Test vs. Gram – Bacteria:

  • Chloramphenicol
  • Kanamycin
  • Nalidixic Acid
  • Nitrofurantoin
  • Sulfa Drugs
  • Tetracycline

Typical Antibiotics Used for Kirby-Bauer Test vs. Gram + Bacteria:

  • Chloramphenicol
  • Erythromycin
  • Novobiocin
  • Penicillin G
  • Streptomycin
  • Tetracycline

What is the purpose of the test?  

The purpose is to see if the microbe is sensitive or resistant to an antibiotic.  This information can be used in the identification of the microbe or in decisions on which antibiotic might be used for the treatment of an infection.

How is antibiotic resistance determined?  

We use the Kirby-Bauer disk diffusion method.  Bacteria are spread across the plate in a thick suspension to support confluent growth across the entire surface – this is called a bacterial “lawn”.  Disks containing antibiotics are added immediately after the agar is inoculated and the plates are placed into the incubator so that the lawn can form.  Meanwhile, the antibiotic diffuses away from the disk, with the area next to the disk having a higher concentration than is found farther away.  In areas where the concentration of antibiotic is high enough to prohibit the bacteria from forming a lawn, a zone of inhibition forms.  After overnight incubation, the lawn has formed and any zones of inhibition represent areas where an antibiotic was effective in preventing growth.  Zones of inhibition are measured (diameter in mm) and compared to standardized results for each antibiotic to determine whether the bacterium was sensitive to the antibiotic (zones of inhibition were larger than the standard size expected) or resistant (zones of inhibition were smaller than the standard size expected).

What medium is used?  

The medium typically used for a Kirby-Bauer Sensitivity Test is Mueller-Hinton agar plates.  This medium supports the growth of most organisms and can be enriched with blood when required to test more fastidious organisms.  

How is the test performed?  

A plate of Mueller Hinton agar is selected and used to create a bacterial lawn.  Antibiotic-impregnated disks are added aseptically to the lawn.  The plates are inverted and incubated overnight, after which zones of inhibition are measured (diameters are measured in millimeters) and compared to standardized charts for interpretation of results..

What reagents are added?  

None.

Performing this test in the VUMIE Online lab

Inoculation of Medium

1. Select Mueller Hinton agar medium.  

2. Complete the process of a tube-to-lawn aseptic transfer using a sterile cotton-tipped applicator (swab) to inoculate the medium.  Forgotten how to do these things?  Watch the “Show Me How To” videos.

3. When you attempt to replace the tube cap and plate lid, you will be prompted to add the antibiotic disks. Do this, and then be sure the cap and lid are replaced.

Incubation of the Inoculated Medium

4. Place the inoculated plate into the 35-37 C incubator.  

5. Press the New Day button to move forward 24 hours.  Forgotten how to do these things?  Watch the “Show Me How To” videos.

Determination of Test Results

6. Incubate this test for 24 hours.  Retrieve the incubated culture. If the test protocol was followed as described above, a lawn of  the culture will have grown and zones of inhibition will be visible revealing areas where growth was prevented due to presence of the antibiotics.

7. Select “Record Results” option when clicking on the plate. A new image will appear showing the same plate with zones marked and disks numbered.

8. A table will appear that contains the list of antibiotics present on the disks and the size ranges for zones representing the standard values for antibiotic resistance (R), intermediate resistance (I), and susceptibility (S). These are specific for each antibiotic. There is also a column that provides the measurements for the zones on your plate produced by the antibiotics you placed there.

9. Compare the sizes of the zones for each of your antibiotics with its standard values in the table. In the final column, provide your interpretation of the results for whether the microbe tested shows resistance, intermediate resistance, or susceptibility to each of the antibiotics.

Updated on July 9, 2023
Acid from glucose

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