The document provides instructions for a microbiology lab on observing bacterial cultures, Gram staining, and microscopy. The key points are: 1. Students will observe cultures grown from the previous lab, Gram stain reference organisms, and observe plates under a dissecting microscope. 2. A Gram stain involves staining a smear with crystal violet and safranin, then observing bacteria as either Gram positive or Gram negative based on retention or loss of the primary stain. 3. When using the light microscope, students will draw and label representative samples of bacteria, noting shape, arrangement, staining method, and total magnification.

1. Ex 3-7: The Gram Stain
Pages 195-202
Today we will do 3 things:
Observe the 2 streak plates and assess the broth culture and
slants from Tuesday for growth patterns
Gram stain some reference organisms
Observe our environmental plates on dissecting scope for
Colony Morphology (Ex. 2-2)
Tools to characterize and identify microorganismsMicroscopic
morphologyColony morphologyCulture
characteristicsBiochemical activities
Each of these is determined by an organism’s genes, and will be
consistent for a bacterial species or isolate
Culture Characteristics in BrothSurface:
Ring, pellicle, none
Sediment:
present, absent
Broth Clarity:

2. Uniform turbidity, flocculent *, clear
Amount of growth:
0 to +3 (none/slight/moderate/abundant)
*
Growth Patterns in Broth pellicle*growth on surfaceflocculent
(clumps)uniform turbidity sediment
1
4
3
2
*
*
*
*
Serratia marcescens. Gram negative, short bacilli that produce
mucoid colonies which have entire margins and umbonate
elevation. Note that there are both red and white colonies
present on this plate. Some strains of S. marcescens produce the
red pigment prodigiosin in response to incubation at 30o C, but
do not do so at 37o C. This is an example of temperature-
regulated phenotypic expression.
Broth: turbid appearance of the broth and the red color present
in both the sediment and pellicle.
Slant: white to red color, smooth, moist growth
Micrococcus luteus. Circular, pinhead colonies which are
convex with entire margins. This gram positive coccus
produces a bright yellow, non-diffusable pigment.

3. Broth: produced granular and pellicle growth
Slant: abundant, opaque, grayish white, smooth, flat, slimy, and
moist.
Escherichia coli. This gram negative, coccobacillus forms
shiny, low convex, mucoid colonies which have entire margins
and are slightly raised. Older colonies often have a darker
center.
Broth: turbid growth with a deposit which disperse upon
shaking.
Slant: off-white in color and showed a spreading growth
pattern: usually it appeared as a cloudy film over the whole
surface of the slant
Staphylococcus saprophyticus A Gram-positive, cocci forms
slightly convex colonies that are opaque, smooth, circular, gray-
white, white to cream colonies.
Broth: uniform turbidity with a fine to slight mucoid deposit.
Slant:
*
Culture Characteristics on a SlantSlants are useful for keeping
stock cultures of bacteriaGrowth occurs on slantThe butt*
allows for diffusion of nutrients to the organisms and diffusion
of waste products away from organismsObserve pattern of

4. growth on the slant:
Note any other unique/obvious characteristics – e.g., color
*
Purpose:To determine appearance of growth on various media
This is consistent for an organism and is useful to characterize
and identify organismsGenetically determined, yet
environmentally influencedCompare organisms to see if they’re
the same or differentCan tell us about their properties, such as
the conditions/media at which they grow
3) Colony Morphology/Culture Characteristics
Colony Morphology/Culture Characteristics
7 Characteristics of Colony Morphology:Shape
MarginElevationSize Color Texture Optical Properties
Shape
Elevation
Margin
Colony Characteristics (p. 64)

5. Size – measure in mm (or a comment)Color – describe the color
(specifically)Texture – typically refers to the colony surface or
its texture when probed with a loopOptical Properties – how it
transmits light (opaque, translucent, transparent)
Dissecting scopes are useful to observe colonies
Colony Characteristics
Summary: Characteristics to observe
On a plate
(as an isolated colony)
Colony
MorphologyShapeMarginElevationSizeColorTextureOptical
properties
On a slant
Growth on slants
Pattern of growth
Anything else that’s obvious or unique
In a broth
Growth in Broth
Surface
Sediment
Broth
Amount of growth
Streak Plate
Last time you created a streak plate from a mixed broth culture
(Namely, E. coli, M. luteus, and Serratia
marcescens)

6. A good streak plate:Is appropriately labeledHas an appropriate
pattern
for streak linesUses the entire plateHas well-isolated coloniesIs
free of contamination
Did you obtain individual colonies?
Observe & critique your streak plate
To get more well-isolated colonies: Use a small amount of
initial bacterial inoculum (in first quadrant).Remember to
flame loop after each quadrant. Don’t go back into previous
quadrant so much (2 or 3 pass throughs). Keep streak
lines moving in forward direction. Use entire plate.
Types of Stains used by MicrobiologistsSimple Stains – a single
dye is used to add contrast, so microbes can be viewed
microscopicallyPositive stainNegative stainDifferential
StainsGram stainAcid-fast stainSpecial Structure StainsCapsule
stainSpore stain Flagellar stain
X
Differential stains detect differences between organismsThe
Gram stain differentiates bacteria into two groups: gram-
positive and gram-negative, based on differences in cell wall
structure.Gram-positive bacteria tend to be killed more easily
by antibiotics like penicillin.Gram-negative bacteria can be
more resistant to antibiotics.
Differential Stains: Gram Stain

7. Will react in a crystal violet dye and will stain dark purple
or violet.
Cell wall is unique because it is composed of several
peptidoglycan layers (multilayered) making it thicker.
Presence of techoic acids
Lacks an LPS (lipopolysachharide) content, periplasmic space
and outer membrane making this group low in both lipoprotein
and lipid composition.
Gram Positive
Normally does not react to a crystal violet dye, stains pinkish
red [decolorized in accepting safranin (a counterstain)]
Thinner peptidoglycan cell wall layer because it is just made of
one single layer as opposed to gram positive’s multi-layered
wall
Lacks teichoic acids, but has a high content of LPS
Has both a periplasmic space and an outer membrane
Has a high amount of lipoprotein content as well as lipids.
Gram Negative
Gram Stain
Start with an air-dried, heat-fixed smear.
primary stain mordant decolorizer counterstain
Crystal violet-iodine complexes form in cellGram-positive

8. bacteriaAlcohol dehydrates & condenses thick peptidoglycan
layerCrystal violet-iodine complexes do not leave
Gram-negative bacteriaAlcohol dissolves outer membrane (LPS)
and leaves holes in thin peptidoglycan
layerCrystal violet-iodine washes out
Gram Stain Mechanism
Differential Stains: Gram Stain
For every Gram stain, observe & record:
Gram reaction
cell shape
cell arrangement
size (if you can)
Gram Stain – Common ProblemsSmears are too
thickDecolorizing too little or too muchOld cells and dead cells
tend to stain Gram negative Because the cell wall is not
intactShould always use fresh cultures
Demonstration of a Gram stainAir-dried, heat-fixed
smearRemember: small amount of organism spread out in a
small drop of water
Gram stain procedure – p 200Decolorization is the most crucial
step
Until run-off is clear
page 200

9. Expectations: Each student should:
Observe & critique your streak plates
Prepare & Gram stain 1 slideSlide: Three smears on slide:
Gram Pos., Mixed, and Gram Neg.
Gram stain procedure – p 200
Observe with oil immersion objective (100 X)
Observe simple stained slides from last lab and colony
morphology
S. Sapro
Or M. luteus
S. marcescens
Or E. coli
Mixed
coccus bacillus spirillum
Bacterial Shape
SinglyPairs: diplococci, diplobacilliClusters:
staphylococciChains: streptococci, streptobacilli
Arrangement
For Next Lab:
Read: Ex 3.9 Capsule Stain Ex 3.10 Endospore StainEx 3.12

10. Flagella Stain
*
*
Scanned by CamScanner
Scanned by CamScanner
Scanned by CamScanner
Lab 2
Ex 3-1 Introduction to Microscopy
Ex 3-5 Simple Stains
Housekeeping ItemsDid you Lysol your benchtop yet? My name
is Professor GiambernardiPronounced “Gim-burr-nardi”Please
introduce yourselves at your tables Seating chart & attendanceIs
everyone finding things on D2L?Remember to purchase sharpies
& gloves

11. Today we will do 3 things:
Observe our cultures inoculated last lab
Become familiar with our microscopes
Perform some simple stains?
Let’s check our cultures from last labNutrient agar platesDid
they grow? How can you tell?Did you see a difference between
unwashed and hand sanitizer?
When finished, discard cultures
in biohazard trash.
Remember to answer questions in
“data sheets” (back of lab section) germane
to the sections we are working on
MicroscopyLight microscope – uses visible light as the energy
source
Brightfield microscopyProduces an image made from light that
passes through a specimenThe background appears
brightObjects appear darker, or might lack contrast
MicroscopyCompound microscope – has two lenses that
magnify the image
Ocular

12. Objectives
The Compound Light Microscope
Light source at bottom
3 lenses produce an image:
The condenser lens focuses & concentrates light to evenly
illuminate the specimen
The objective lens collects light, magnifies, and forms an image
that you see with your eyes.
The ocular lens further magnifies the image formed by the
objective lens.
*
Concept #1: MagnificationMagnification = the size of the
imageFour different objective lenses:
scanning low power high power oil immersion
4X 10X 40X 100X
Ocular lens: 10XTotal magnification = (objective mag) x
(ocular mag)Parfocal = object in focus under 1 objective will be
in focus under all objectivesParacentered = each objective will
successively focus on the center of the previous field of view
MagnificationField diameter = the actual diameter of the area
viewed, using those particular lenses Units will be a distance
(mm, µm) As magnification increases, field diameter
decreases
Objective Field diameter

13. scanning 4.5 mm
low power 1.8 mm
high power 0.45 mm
oil immersion 0.18 mm
MagnificationDepth of Focus = the amount of vertical space
that appears in focus, using those particular lensesSimilar to
depth of field in photography
As magnification increases, depth of focus _____?______
decreases
Concept #2: ResolutionResolution – the clarity of the image
Resolution & Car Headlights
UNRESOLVED PARTIALLY RESOLVED
RESOLVED
Concept #2: ResolutionLimit of resolution (resolving
power)How far apart 2 points must be to be distinguished as
separate Indicates the size of the smallest object that can be
clearly observed with those lensesThe limit of resolution is

14. decreased as wavelength is decreased.
High magnification without high resolution is “empty.”
Magnification & Resolution
*
Limit of ResolutionAs magnification increases, the limit of
resolution gets smaller
For example, Eye 0.1 mm (millimeters)Light
Microscope
3.0 nm (nanometers)Transmission EM 0.2 nm
(nanometers)
Question about Resolution:We just said the limit of resolution
Will we be able to see this bacterium using our microscope?
Why or why not?
Concept #3: Working DistanceThe amount of clearance
between the slide and the bottom of the objective lensAs
magnification increases, working distance _________Don’t

15. crash your objective lens into the slide!
decreases
Concept #3: Working DistanceThe amount of clearance
between the slide and the bottom of the objective lens
4X
40X
10X
Concept #4: RefractionRefraction – bending of light as it
passes through an objectRefractive Index – a measure of the
light-bending ability of a medium
Concept #4: RefractionOil minimizes refraction to capture
more light with the objective lens
Your microscopeLet me demonstrate proper carrying of the
microscopeCarrying your microscope to your benchYour seat #

16. = your microscope #Let’s get our microscopes (located in
cabinet next to you)
Microscope parts and their functionOcular – provides
magnificationObjective – provides magnificationStage – holds
the specimen; enables movement of the specimenCondenser –
concentrates or focuses light to illuminate the specimenIris
diaphragm – changes the size of the cone of light, thus adjusting
the amount of lightCoarse focus knob – raises & lowers the
stage in larger increments, to adjust the focusUse ONLY with
scanning & low power objectivesFine focus knob – raises &
lowers the stage in smaller increments, to adjust the focus
Stage – holds the specimen;
enables movement of the specimen
Microscope parts and their function
Stage
Stage clip
Tips for Today – Using Immersion OilUse oil only with the oil
immersion objective (100X)Get the specimen in focus under any
lower power (4X or 10X) objectiveSwing that objective out of
the way, add a small drop of oil right on the specimen, and
swing the oil immersion objective into place-oil will contact
100X objectiveDon’t move the stage down to add oilUse only
the fine focus knob with oil immersionClean up all oil with lens
paperObjective, stage, specimen, etc.

17. Tips for Today - DrawingsPurpose of drawings is to cause you
to carefully observe what you’re seeing Draw a representative
sample This is not art class
Every drawing must be labeled with the name of the specimen
and the total magnification For example: yeast suspension,
100X
Tips for Today – Returning your microscope to the cabinet
Remove slideClean off all oil, using lens paperEngage the
lowest power objective lens (4X)Wrap cord neatly around the
baseReplace dust coverPlace scope carefully on the shelf, with
oculars spun around and arm outward
Expectations: work as individualsExamine a slide of letter
“e”Mount so “e” appears correctly
as you see the slide on the stage
Examine prepared slide of colored threadsExamine using low
power (10X) & high power (40X) objectives
Examine a prepared slide of bacteriaExamine using scanning,
low power, & high power objectives
e
Why make smears and stain bacteria?Looking at a bacterium

18. under the microscope may be one of the first methods used to
identify the cause of an infectionDirect exam of specimenAfter
culture of specimen
What’s the limitation of brightfield microscopy? Bacterial cells
are very small and lack contrast. With brightfield microscopy,
a stain is used to add contrast and make them visible.
Preparing Specimens for
Light Microscopy – The SmearA smear is a thin film of
microbes on a slide.From broth: A loopful of broth cultureFrom
slant/plate: A small amount of a colony is added to a very small
drop of waterLet the slide air-dryA smear is heat-fixed in order
to: attach the microbes to the slidekill the microbesincrease
stain penetration.
Types of Stains used by MicrobiologistsSimple Stains – a single
dye is used to add contrast, so microbes can be viewed
microscopicallyPositive stainNegative stainDifferential
StainsGram stainAcid-fast stainSpecial Structure StainsCapsule
stainSpore stain Flagellar stain
Simple StainsPositive stainBasic (positively-charged) dye
interacts with negatively-charged bacterium.Colored bacterium
appears in a bright field. Negative stainAcidic (negatively-
charged) dye is repelled by negatively-charged bacterium.
Stains background and leaves cells unstained.

19. Positive and Negative Staining
Positive stain
Negative stain
Making a Bacterial Smear (Ex. 3.5, pg. 186-187)
When observing a stained smear, you should always observe &
record: Shape Arrangement Size (a comment, if you can)Don’t
forget to label every drawing
What information in the label? Specimen, total mag, staining
procedureReminder: total magnification =
ocular (10X) X objective (4X or 10X or 40X or 100X)
Simple stains
coccus bacillus spirillum
Bacterial Shape
SinglyPairs: diplococci, diplobacilliClusters:
staphylococciChains: streptococci, streptobacilli
Arrangement
Stay organized with your label & observations
Tip for Today: Drawings
Name of bacterium
1000X

20. Simple stain with safranin
bacillus in
singles
singles
bacillus
1000X
Simple stain
Name of bacterium
safranin
For Next Lab:Read: Ex 2-2 Colony Morphology and
Ex 1-3 Aseptic Transfers & Inoculation Methods (pgs. 29 - 48)
Welcome to General Microbiology Lab
Biol 2421
IntroductionsMy name is Professor Troy
GiambernardiPronounced “Gim-burr-nardi”Please introduce
yourself to the students at your table. Name, program of study,
how long you’ve been a student at Lone Star College, how &
how far you come to get hereClass list: Please tell me your
name, using the name you would like to be called Some
information, please: Student Information
SheetNext lab, I’ll circulate a seating chart

21. Syllabus and Schedule are posted on Desire 2 Learn (D2L)Class
& Lab will use the same D2L siteLab SyllabusLab Schedule
Safety ConsiderationsRespect live microorganisms!Please let
me know later if you are immunocompromised or
immunosuppressedNo food or drink in the laboratoryLab coat,
smock, or old shirt is highly recommendedClose-toed shoes are
highly recommendedYou are responsible for your own
GlovesMust cover any cuts/open sores with gloves or band-
aidOther safety itemsRaise any safety concerns or questions
Procedure for Disinfecting a Lab SpillCover generously with
Lysol®Lay paper towels on topLet sit for 5-10 minutesClean it
upWear gloves and/or use brush & dustpanBe cautious of
broken glassDiscard into biohazard trash
Basic Rules & Procedures for Working in the Biology
LaboratoryPlease sign Basic Rules and Procedures Sheet
circulating around the labPut on the instructors table up frontA
duplicate copy is at the back of the class syllabus on D2L
Before you come to labPlease look at scheduled activities on the
lab schedule in the syllabusPlease read the lab exercises in the
Lab Manual Microbiology Laboratory Theory & Application,
4th edition; Leboffe & PiercePlease bring the lab manual with
you to lab.

22. Summary of how lab runs:Put away gum, drinks, cell phones,
etc.Disinfect bench with Lysol.Listen to short introduction –
take notes.Do the lab exercise(s).Complete “Data Sheet”
questions at the back of each section of your lab manual
germane to the Exercise(s) we are working on.These will be
collected along with the posted Lab Packet questions for
grading.Disinfect lab bench.Wash hands before you leave.
Exercise 1-1
Glo Germ Hand Wash
Glo Germ Hand WashingPurpose:To assess proper hand washing
techniquesTo keep the lab environment from becoming
contaminatedTo keep us from becoming infected by
our culturesFollow protocol from instructor
Glo Germ Demo
Bathroom Hygiene Myth | MythBusters
Toilet Germs Reach Your Tootbrush?
Inoculating Petri Plates from Environment Cultures
Step 1: Obtain a sterile cotton swab, sterile saline, and one agar
plate.
Step 2: Choose a surface to sample.
Step 3: Label the bottom of your plate with sample name, your

23. name or initials, and date.
Step 4: Remove sterile swab, wet swab with saline, and sample
surface of interest with wetted swab.
Inoculating Petri Plates
Step 5: Holding the petri dish lid at an 30°-45° angle, work the
contaminated swab from the outside of the plate toward the
center in a zig-zag pattern that covers approximately 25% of the
plate surface (think pie or pizza slice!).
Inoculating Petri Plates
Step 6: Turn the petri plate 90° to the right, dragging the
inoculation loop through the last section of the plate, moving
from the outside to the inside in a zig-zag motion.
Step 7: Repeat this process twice more until the entire plate
surface is covered.
NOTE: If you are trying to isolate individual colonies, each turn
of the dish will give you fewer microbes so that you can
distinguish individual colonies.
If you streak your plates correctly, the pattern should look like
this on your plate.
*
If you streak your plates correctly, the pattern should look like
this on your plate.

24. Inoculating Petri Plates
Step 8: Replace plate lid and invert the plate for incubation at
37°C.
Plates are inverted (upside down) to avoid condensation spilling
onto your cultures.
Ex 3.9 Capsule Stain
Ex 3.10 Endospore Stain
Ex 3.12 Flagella Stain
Week 3
Today we’re doing:
Differential stains on some reference organisms:
Capsule stain
Endospore stain
Flagella stain
Finish Gram Staining
Differential Stains: Endospore StainDifferentiates bacteria
based on ability to form endospores or notCan often visualize
the presence, shape, and location of spores within a vegetative
cellSpores are resistant forms; heat is needed to drive the stain
into the sporesSpore-forming bacteria include Bacillus species

25. and Clostridium species
The Genus ClostridiumGram-positive,
spore-forming rodsAnaerobes> 100 speciesPathogens: C. tetani
- tetanusC. botulinum - botulismC. perfringens – gas
gangreneC. difficile - antibiotic-associated
colitisNonpathogens:Live in large bowel as commensals
Clostridium tetani
Clostridium difficile
Spore stain (p 217)Endospores are resistant; stain does not
readily penetrate sporesSpores will stain with Malachite Green
(primary stain) and heatDecolorize with water; vegetative cells
will lose the malachite greenCounterstain with safraninSpores
will appear _________ and vegetative cells will appear
________.
blue-green
red/pink
Spore
Stain
Spores will appear _________ and vegetative cells will appear
________.
blue-green
red/pink

26. The Genus BacillusGram-positive,
spore- athogens: B.
anthracis – anthraxB. cereus – food poisoningNonpathogens:B.
subtilis – source of enzymesB. polymyxa – source of antibiotics
Bacillus anthracis
Spore Stain – think about itWhen do spore-forming bacteria
undergo sporulation?Are spores likely to be observed in fresh
cultures or on a rich medium? If spores are not observed:it
might mean that spores weren’t produced under the conditions
of growth or, it might mean this organism is not a spore-former
Expectations – Work as individuals
Spore stain – p. 217Prepare a smear on a slide each for:
Bacillus subtilis and Bacillus cereus
Observe slides using 100X oil immersion objective
Observe Flagella stained slides on instructors table at front
After that, continue with your prepared simple and Gram stained
slides
p. 217
Steam w/ Malachite Green stain for 10-15 min
Differential Stains: Capsule StainDifferentiates bacteria based
on ability to produce a large capsule or very little/no capsule
Capsule is one kind of glycocalyxUsually composed of

27. polysaccharideProtects cell from drying and from host defenses
(anti-phagocytic)Caution: Capsules are somewhat fragile:
Destroyed by heat-fixingCan be washed awayStaining for a
capsule is useful for identification and classification
Capsule stain (p 212)Small amount of bacteria is suspended in
Congo red – an acidic dye (Ignore the serum)Acidic dye will
stain/not stain the bacterial cellDraw out the drop to create a
smearDo not heat fix your smear! Let it air dryFlood the smear
with Maneval’s stainBasic dye will stain/not stain the bacterial
cellGently rinse with water. Observe using oil immersion lens
(100X)Distinguish between a tiny halo (from cell shrinkage)
and a true capsule.
(p 212)
Capsule
Stain
FlagellaFlagella allow for motilityBacterial flagella rotate
rather than
whipArrangementsMonotrichousPeritrichousLophotrichousAmp
hitrichousPresence/absence, number & placement of flagella are
useful for identification & classification
Monotrichous
Amphitrichous

28. Lophotrichous
Peritrichous
A
B
C
D
How would you determine if a bacterium has flagella? Look for
flagella microscopically But hard to see with the light
microscopeLook for motility an indirect assessment of the
presence of flagellaSo, if a cell is motile, it must have
flagellaWe’ll look for motility using a wet mount
Bacterial Motility
Videoclip2 of Bacterial Motility
Videoclip1 of Different Types of Bacterial Motility
Motility Test MediumA semisolid medium, containing a reduced
amount of agarInoculate vertically, using an inoculating
needleMotility is apparent if organisms grow and spread away
from the line of inoculationNonmotile organisms will only grow
at the line of inoculation
Motility Test Medium
+ – – +

29. Disinfectants & Antiseptics
Antibacterial Susceptibility Test
Ex 2-14 Disinfectants & AntisepticsEx 7-3 Antimicrobial
Susceptibility Test
Chemical Agents - General PrinciplesSterilization
– killing or removal of all life forms
Disinfection/Antisepsis – removal of pathogens; reduction in
the number of organisms, so they pose no danger of
diseaseDisinfectant – chemical agent used on an
inanimate object
Antiseptic – chemical agent used externally or topically on
living tissue
*
Effect of Chemical Agents on Bacteria
There are many different methods to assess effects of chemical
agents on bacteria
Laboratory methods – Assess the properties of agents under
laboratory conditionsE.g., methods to evaluate antibacterial
potencyE.g., methods to evaluate –cidal or –static activityIn-use
methods - Assess the effectiveness of agents under conditions
similar to actual useE.g., methods to evaluate performance for

30. particular uses or in real-life situationsE.g., methods to compare
performance of 2 agents
Chemical Agents – Tube Dilution ProcedureDetermining the
antibacterial potency of an agent against Staphylococcus aureus
(S. aureus)
or Escherichia coli (E. coli)
Agents: Isopropanol, H2O2, Bleach, or Lysol
at various concentrations Inoculated with S. aureus or E. coli
by swab technique with fresh broth culture
Determine the lowest concentration of that agent that inhibits
growth of the test organism
1.
4.
2.
3.
5.
6.
8.
7.
1 min.
10 min.
dry for 10 min.
Effect of Chemical Agents
So which procedure is a laboratory method?
Beads procedure

31. Which procedure is an “in-use” method?
Fingertip antisepsis procedure
Chemical AgentsActive ingredient should be noted on the
containerClass = the general group to which the active
ingredient belongs:
Product ClassIsopropanol
AlcoholsHydrogen Peroxide Oxidizer(H2O2)Bleach
Oxidizer
(Sodium Hypochlorite)Lysol Quaternary
Ammonium
Compounds
Effectiveness
(Lowest Concentration of Reagent
Needed)OrganismBleachH2O2IsopropanolLysolS. aureusE. coli
Antibacterial Susceptibility
Ex 7-3 Antimicrobial Susceptibility Test

32. Why Study Antibacterial Susceptibility Testing?Clinical labs
routinely perform “culture & sensitivity”
testingIdeally, effective antibiotic treatment is directed against
the specific pathogen infecting the patientSome strains of a
species have acquired resistance to antibacterial agents
*
Antibacterial AgentsSpectrum of activityBroad spectrum –
Active against a broad array of bacteria, including both
Gram(+) & Gram(-) organismsNarrow spectrum – Active against
a limited range of organisms
The Sites of Activity in a Bacterial Cell for Various Antibiotics
Chloramphenicol inhibit protein synthesis
Ciprofloxacin: Iinhibit DNA gyrase
Trimethoprim: inhibits bacterial DNA synthesis
Penicillin: inhibit the formation of
peptidoglycan cross-links in the bacterial
cell wall
Kirby-Bauer method for determining antibacterial
susceptibilityDisc-diffusion method
Antibiotic discs Contain defined amount of a specific

33. antimicrobial agentAgent diffuses out of disc radially, into the
mediumForms a concentration gradient in the medium
Zone of inhibition Zone size determined by concentration of the
agent that inhibits bacterial growth
Assessing Antibacterial Activity
How to get from a zone of inhibition to effectiveness (or lack of
effectiveness)?
Measure zone of inhibitionBigger zone is betterZone size alone
doesn’t determine susceptibility or resistance
Must considerFactors associated with the test
methodConcentration of drug in the body after a usual dose
*
Kirby-Bauer method for determining antibacterial
susceptibility(Mueller-Hinton Agar used)non-
selective, non-differential medium (almost all organisms plated
will grow)has a few properties that make it excellent for
antibiotic usecontains starch. Starch is known to absorb toxins
released from bacteria, so that they cannot interfere with the
antibiotics. it is a loose agar, which allows for better diffusion
of the antibiotics than most other plates. A better diffusion
leads to a truer zone of inhibition.
Interpretive Criteria
Interpretive criteria convert zone size (mm) to 1 of the
following categories:S = susceptible; pathogen with that zone

34. size will likely be killed/inhibited by the concentration of drug
in the bloodstream provided by the usual dose of that agentThis
agent should provide effective therapyR = resistant; pathogen
with that zone size will NOT likely be killed/inhibited …Select
a different agent for therapyI = intermediate; provides a buffer
zone to prevent small differences in the test from having a
major impact on interpretationSelect a different agent for
therapy
Antibacterial Susceptibility – Day 2Measure diameter of zone of
inhibition with ruler (in mm)Record data for all 4 agents
& 2 different organisms (Worksheet 7-3 pg. 661)
Use interpretive criteria to convert zone size (mm) to a category
(page 271): S = susceptible I = intermediate R = resistant
Zone diameter (mm)
Interpretive CriteriaConvert zone size (mm) to S, I, or R
AgentZone size (mm)Susceptible
IntermediateResistant Chloramphenicol - C30
Enterobacteriaceae & staphylococcus
>18
13 - 17
<12Ciprofloxacin - CIP Enterobacteriaceae &
staphylococcus>2116 - 20<15Trimethoprim – TMP5
Enterobacteriaceae & staphylococcus>1611 - 15<10Penicillin –
P10 Staphylococcus>29<28

35. Expectations for Today: Work as table-teams
2 Mueller-Hinton Agar plates & 1 pkg swabs per table
Inoculate each plate with 1 organism:
Swab in 3 directions for confluent growth
Staphylococcus aureus Escherichia coli
Apply 4 different discs to each plate
Chloramphenicol - C30, Ciprofloxacin – CIP, Trimethoprim –
TMP5, Penicillin – P10
Use flamed forceps; tap disc gently for good contact with
agar
Incubate in the inverted position at 37°C
Disc will not fall off, even upside down in the plate
We will observe/interpret results next lab
C30
CIP
TMP5
P10

36. Disinfectants & Antiseptics
Antibacterial Susceptibility Test
Day 2: Interpretation of Results
Ex 2-14 Disinfectants & Antiseptics
Ex 7-3 Antimicrobial Susceptibility Test
Expectations for TodayRead & interpret antibacterial
susceptibility testMeasure zone size (mm)Use interpretive
criteria to convert zone size to S,I,R
Summary of Class Data
AgentS. aureusE. coliChloramphenicol - C30 Table 1 =
Table 2 =
Table 3 =
Table 4 = Table 1 =
Table 2 =
Table 3 =
Table 4 = Ciprofloxacin - CIP Table 1 =
Table 2 =
Table 3 =
Table 4 = Table 1 =
Table 2 =
Table 3 =
Table 4 = Trimethoprim – TMP5 Table 1 =

37. Table 2 =
Table 3 =
Table 4 = Table 1 =
Table 2 =
Table 3 =
Table 4 = Penicillin – P10 Table 1 =
Table 2 =
Table 3 =
Table 4 = Table 1 =
Table 2 =
Table 3 =
Table 4 =
Lab 1
Aseptic Transfers & Inoculation Methods (Ex 1-3)
Streak Plate Method (Ex 1-4)
Aseptic TechniqueAseptic = free of contaminationPurpose:To
keep our cultures from becoming contaminatedTo keep the lab
environment from becoming contaminatedTo keep us from

38. becoming infected by our cultures
Labeling culturesAlways include:The name of the organism or
specimenYour name or initialsThe date inoculatedFor
broths/slants, label so you can still see growthFor plates, label
on the bottom, the side with the growth mediumLabel before
you inoculate
Sources of Contamination
From our culture’s point of view: usthe airobjects & surfaces in
the environment
From our point of view: the culture
Materials we’ll be using todayCulture MediaBroths (1 per
student)Slants (1 per student)Plates (2 per student)Inoculating
LoopBunsen BurnerFlint Striker
Demonstration of slant to broth transfer
(See Ex1-3, p. 32-37 in lab manual)Tube of broth &
labelingAseptic techniqueInoculumTransfer to fresh
brothIncubation at 37°C
Demonstration of slant to slant transferSlant & labelingAseptic
techniqueInoculumTransfer to fresh slantIncubation at 37°C

39. Streak Plate Method
Purpose: To check or establish the purity of a culturePure
culture = contains a single speciesMixed culture = contains
more than 1 species
Principle: Organisms are diluted out over the surface of a
growth medium, so individual cells will grow to form visible
coloniesColony = a visible mass of cells1 cell = 1 colony-
forming unit (CFU); The assumption is that 1 cell grows to form
1 colony
Streak Plate Method (see p. 43)
Quadrant Streak Method
Heat loop
Inoculate culture
Streak
1st quadrant
A good streak plateIs appropriately labeledHas appropriate
pattern for streak linesUses the entire plateHas well-isolated
coloniesIs free of contaminationOne colony type
Demonstration of Streak Plate MethodAgar plate &
labelingAseptic techniqueInoculumStreak patternIncubation
(inverted) at 37°C

40. For your review: Video of aseptic technique (2 min)Video of
streak plate method (1½ min)
Expectations: Work as individualsAseptically transfer from
stock slant culture to a fresh broth and to a fresh slant – use “S.
marcescens ” or “M. luteus” or
“E. coli” or “S. sapro”Label appropriately with the organism,
your name or initials, & date
Aseptically Streak 1 plate using the quadrant methodUse
“culture used above”Label appropriatelyIncubate in plate in
inverted position
Aseptically Streak a 2nd plate using the quadrant method Use
“mixed culture” Label appropriatelyIncubate in plate in inverted
positionPut all your cultures into the incubator (37°C), on the
shelf for this section
Expectations: Work as individualsAfter inoculations are
complete, Work on Simple Stain from last weekEx. 3.5, pg.
186-187 Making a Bacterial Smear and Simple Stain procedure.
Gather around for demonstration
(use real inoculum)Broth to broth transferPlate to slant
transferStreak plate method
NAME:

41. Section/Time:
Microbiology Lab Packet #2
Think of these lab packets as homework for the lab part of the
class. They are designed to help prepare you for the
departmental lab exams. To answer these questions you will
need to use both the lab workbook as well as your text book. In
addition to these questions, you will need to turn in the signed
data sheets from your workbook.
19. Describe the molecular basis behind Gram staining. What
part of the bacteria is stained with the primary stain, what part
is stained with the counter stain? Why do we wash alcohol over
the slide before the counter stain?
18. Describe in great detail the steps in performing a Gram
stain. Include incubation times. Define primary stain and
counter stain. In the Gram stain, what dye is primary stain,
which is the counter stain?
Special Stains (Capsule Ex. 3.9, Endospore Ex. 3-10, Flagella
Ex. 3.12)
21. What is the stain is shown in the picture below? What is
stained green, what is stained red?
22. Describe in great detail the steps in performing an
endospore stain. Include incubation times.
20. What does it mean to be Gram positive, Gram negative?

42. 23. Label the different flagellum arrangements seen below.
24. Describe the negative staining technique and determine
when it is used.
Colony Morphology Ex 2.2
25. We can learn some information about bacteria by just
looking at its growth on an agar plate. What is a small isolated
growth of bacteria on an agar plate called and what can it tell us
about the bacteria?
Micro lab packet 2
Kirby-Bauer Method Ex 7.3
26. Describe in great detail the technique used to determine
bacteria sensitivity to antibiotics?
27. What is the type of media used in the Kirby-Bauer test and
why is it used?
Describe what is observed for a bacteria that is sensitive to
antibiotics in a Kirby-Bauer test, and to a bacteria that is not
sensitive to antibiotics.
28. How does the antibiotic get from the disk into the agar?
Does the agar have an antibiotic beyond the zone of inhibition?
29. Define the MIC and explain how it is used to determine if a
bacteria is resistant or sensitive to the antibiotic.
Is the presence of a clear zone around the disc enough evidence
to determine antibiotic resistance?
Micro lab packet 2
Disinfectants Ex 2.14
30. Compare your results with the class data.

43. Which germicide was most effective and at what concentration?
Which was the least effective? Defend your choices.
31. Which organism seemed to be most resistant to the
germicides?
32. What purpose did the four controls serve? Be specific.
Include Completed Data sheets for:
1. Data Sheet 3-7 (pages 201-202) Sketch & id whether Gram +
or Gram – and Questions 1-2
2. Data Sheet 3-9 (pages 213-214) Sketch capsuled stained
bacteria; Questions 1 & 2
3. Data Sheet 3-10 (pages 219-220) Sketch Endospore Stained
bacteria; Questions 1-4
4. Data Sheet 3-12 (pages 227-228) Sketch Flagella Stained
bacteria; Questions 1 & 3
5. Data Sheet 2-14 (pages 139-140) Questions 1-3
6. Data Sheet 7-3 (pages 535-536) Questions 1-7
Gram Staining 3.7
33. What is the difference between disinfectants and
antiseptics?