Kirby-Bauer test and
Endospore Staining
Kirby-Bauer or Disk Diffusion Test
• Designed to test the effectiveness of antibiotic
agents and chemicals at inhibiting bacterial
• Uses Mueller-Hinton agar, allows agent to
diffuse freely from disk
• Primary test preformed in clinical laboratories
with strict quality control

Petri plates with isolated colonies
Petri plate with Mueller-Hinton agar
Sterile cotton swab
Sterile DI water
Dispenser, antimicrobial disks and/or disks
impregnated with other agents
• Forceps
• Alcohol
• Ruler
• Soak tip of sterile cotton swap in sterile water
• Pick up single colony from designated plate
• Swab entire target plate with cotton swab, make
sure to crisscross the plate multiple times from
multiple directions to ensure full coverage
• Allow plate to sit for 5 minutes
• Using sterile forceps (via microincinerator) Place
impregnated disks evenly spaced around petri
• Gently tap the disk to ensure full contact with
• Write a code for each of the disks on the bottom
of the petri plate to reference results and records
• Invert and incubate
• Use the table below as an example to record
Bacterium 1
Bacterium 2
*S – susceptible; I – Intermediate; R – Resistant
Bacterium 3
Interpretation of Zones
Report Format

Title: Top of 1st page

Authors: list authors in alphabetical order

Abstract: a brief summary of the experiment and results

Material and Methods: Describe the actions taken in each
step, formulas used both math and media.

Break down by steps i.e. media prep, inoculation, cell counts

Include tables or figures as needed to summarize data sets like
nutrient targets and masses used.
Cross-Reference: When including tables and figures make
sure to cross reference them in the document where they
are mentioned i.e. “the target concentrations of the
media can be found in Table 1”, or nutrient mass were
weight out (Table 1) on a MT SI-114 balance”
Report Format

Results: Keep same formatting as material and
methods, but describe the results from each step.

Do not make conclusions or explain why the result was

Include cross referenced figures/tables at end of section
Conclusions: Explain why your results came out as they
did. Make observation referencing your results, and
hypothesize what could be done to change your results

If odd things need to be explained at each step then break
down by sections

If everything went according to materials and methods then
a paragraph is fine
References: If any references are used as a descriptor
for a technique used or to compare results include
them here.
Schaeffer-Fulton Endospore Staining
• Spores are resistant due to the protein keratin
that makes up the outerlayer
• Keratin is also resistant to stains
• Uses Malachite green to stain the spore
• Malachite green is water soluble and has low
affinity for cellular material i.e. washes out of
bacterial cell walls but not spore cell walls
• Prepare a smear of
Bacillus and allow to air
• Heat fix the smear by
holding back side of slide
against microincinerator
• Flood the heat smear
with malachite green and
allow to stand for 5
minutes then apply heat
for 10 second
• Wash smear with gentle
stream of water
• Counterstain with
safranin for 45 seconds

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