Bacteriology. laboratory organization by Dr.T.V.Rao MD

1. Bacteriology Laboratory
Organization and Skills
Dr.T.V.Rao MD
11/13/2023 Dr.T.V.Rao MD 1

2. Bacteriology Laboratory
Bacteriology
Laboratory makes
the Backbone of
any Hospital and
without which no
hospital can
function to the
Minimal needs, All
the Microbiologists
and Lab
professionals need
the basic skills and
safety for
effective
functioning of
services

3. Before staring, be familiar with Normal
pathogenic, and opportunistic pathogens
• Normal
Flora
• Opportunis
tic
Pathogens
• Pathogens

4. Microbiology and the Role of
the Microbiologists
• Microbiology – study of microorganisms
(simple forms of life visible only with a
microscope)
• Microorganisms
–Normal flora
–Pathogenic

5. Medical technicians can be
Assists physician / Microbiologists
Obtains specimens
Prepares specimens for direct examination
Prepares specimens for transportation to
reference laboratory
If office has a POL, performs microbiologic
procedures
Microbiology and the Role of
the Medical Technicians

6. Classification and Naming of
Microorganisms
• Classification by structure
–Subcellular – DNA or RNA surrounded
by a protein coat – viruses
–Prokaryotic – simple cell structure
with no nucleus or organelles –
bacteria
–Eukaryotic – complex cell structure
with nucleus and specialized
organelles – protozoans, fungi,
parasites
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7. • Special groups
– Mycobacteria – bacilli
with a cell wall that
differs from most
bacteria
– Rickettsia
• Very small
• Live and grow within
other living organisms
such as mites and ticks
– Chlamydia
• Cell wall structure
differs from other
bacteria
• Live and grow within
other living cells
– Mycoplasmas –
completely lack the
rigid cell wall
Bacteria: Classification and
Identification (cont.)
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8. Bacteria
Single-celled prokaryotic organisms
Reproduce rapidly
Classification
Shape
Ability to retain dyes
Ability to grow
with / without air
Biochemical reactions
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9. • Ability to retain certain dyes
– Gram’s stain
– Acid-fast stain
• Ability to grow in presence or absence of air
– Aerobes – grow best in the presence of oxygen
– Anaerobes – grow best in the absence of
oxygen
• Biochemical reactions
Bacteria: Classification and
Identification (cont.)
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10. Bacteria: Classification and
Identification
• Shape
–Coccus – spherical, round, or ovoid
–Bacillus – rod-shaped
–Spirillum – spiral-shaped
–Vibrio – comma-shaped
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11. All Microbiologists should be
familiar with :
• Clinically significant bacteria
–Morphological characteristics
–Biochemical characteristics
–Signs and symptoms they cause in the host
they are infecting
–Virulence factors
–Pathophysiology of infection

12. How Infections Are Diagnosed
 Steps to diagnosis and treatment
1. Examine the patient
Presumptive diagnosis
May or may not need additional tests
2. Obtain specimen(s)
Label properly
Include presumptive diagnosis
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13. How Infections Are Diagnosed
(cont.)
3. Examine specimen directly
• Wet mount
• Smear
4. Culture specimen
Culture medium – contains nutrients
Examine culture visually and
microscopically

14. Before starting the work ..
Different media are used to culture microorganisms,
be certain that you are using the appropriate media
for your organism.
Always use sterile technique to prevent
contamination.
Choose the type of media (liquid or plate) appropriate
for your investigation or application.
Sterile liquid culture tubes and media plates can
be prepared in advance and stored in the
refrigerator for later use (2 weeks for liquid
culture tubes, 2 months for media plates).

15. Before starting work …
Liquid culture tubes, solid slant tubes, and petri
plates can be used to culture microbes.
Media and lab materials should be sterilized prior
to use; an autoclave or a pressure cooker can be
used in the sterilization process.
Serial dilution and plate count
techniques are used to estimate
microbial populations from
environmental or commercial
cultures.

16. Specimen Collection
Must be collected
correctly
If not, may not grow in
culture
Contaminants may be
mistakenly identified
Patient may receive
incorrect or harmful
therapy
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17. Specimen Collection (cont.)
• Devices
– Use appropriate collection
device or specimen container
– Sterile swabs – absorbent
material on the tip
• Collection and transporting systems
– Sterile, self-contained
– Transport medium
– Aerobic or anaerobic

18. Specimen Collection: Guidelines
Avoid causing harm,
discomfort, or undue
embarrassment
Collect from
appropriate site
Obtain specimen at
correct time
Use appropriate devices
Obtain sufficient
quantity of specimen
Obtain specimen prior
to the start of
antimicrobial therapy
Label correctly
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19. Specimen Collection (cont.)
Throat culture specimens
Swab back of throat in the
area of the tonsils
Avoid touching any
structures in the mouth
Prepare culture plate or
prepare correctly for
transport to laboratory

20. Specimen Collection (cont.)
Urine specimen
Clean-catch
midstream to
minimize
contaminants
Process within 60
minutes or
refrigerate
Sputum
specimen
Specimen from
lungs
Avoid
contaminating
specimen with
saliva
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21. Specimen Collection (cont.)
Wound specimen
Swab wound or
lesion
Do not touch outside
of wound
Stool Specimens
Technique varies
Bacterial infection
Protozoal or parasitic
infection
Instruct patient in
correct collection
procedure
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22. Transporting Specimens to an
Outside Laboratory
 Many offices send cultures to an outside lab
 Three main objectives
 Follow proper collection
procedures and proper
collection device
 Prevent deterioration of
specimen
 Protect anyone handling
specimen
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23. Direct Examination of Specimens
Enables physician to initiate treatment immediately
Wet mounts
Nacl mixed with
specimen of glass slide
Presence of pathogen
and movement of
microorganism
Potassium hydroxide
(KOH) mounts
Used if a fungal
infection of the skin,
nails, or hair is
suspected
KOH dissolves keratin
that can mask
presence of a fungus
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24. Preparation and Examination of
Stained Specimens
 Quick, tentative
diagnosis
 Differentiation
between types of
infections
• Gram’s stain
– Moderate-
complexity test
– Bacteria either retain
or lose purple color
• Gram-positive
bacteria
• Gram-negative
bacteria
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25. Procedure for Making a ‘Smear’
• Using aseptic technique remove a colony from a
plate or cells from your slant. Be carefully to
gently touch the surface of your culture with the
inoculating loop.
• Make a circular motion in the middle of the circle
to spread the cells equally in this region of the
slide
• Add a drop of water in the middle
• Mix again
• Let Air dry
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26. Making a Smear
• Wash the glass slide thoroughly
with soap and water then rinse
with 95% alcohol to sterilize.
• 2. Allow the slide to dry properly.
• 3. Pass the clean slide over a
flame with its face down to
further sterilize it. (Make sure to
hold it by its edge)
• 4. Draw a small circle on
the slide so you can put
your bacteria on the back of
the marked area.
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27. Smear Preparation
• Smear Preparation
• Only a small amount of
bacterial culture should be
used.
• Thick smear causes
overcrowding of a large
number of cells.
• Two different media require
two different techniques
• Liquid Medium/ Broth Culture
• 1. Take the loop and hold it
in the flame at 45o until it
turns red. Your loop is
inoculated now. Let it cool for
a few minutes.
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28. Procedure for Making a ‘Smear’
• Run the slide through
the flame until the slide
is warm ( The frosted
side should be
down) This fixes the
bacteria to the slide
• Let the slide cool
• Place in the metal tray
or in the rack
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29. Media Types
•General
Purpose Media
•Enriched
Media
•Selective
Media
•Differential
Media
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30. Culturing Microorganisms
• There are two basic culture
techniques used in microbiology:
1. Liquid culture: bacteria, algae, and
some fungi can be reared in culture
tubes (test tubes) in a liquid medium.
 Liquid medium is best when you want to
rapidly increase the concentration of
the organism or when you want to grow
motile cells.
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31. Culturing Microorganisms
• There are two basic culture techniques
used in microbiology:
2. Culture Plates: Liquid medium is solidified
using agar (Agarose) and poured as a thin
layer in the bottom of a culture dish (also
sometimes called petri plate)
 Culture plates are used when you want to test (1)
antibiotic sensitivity, (2) estimate culture
concentrations from environmental samples, or
(3) isolate individual colonies from environmental
samples.
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32. Culturing Specimens in the
Laboratory
• More common to send
specimens for culture to
outside labs
• Culturing involves placing a sample of
specimen on a culture medium
– Medium – nutrients
– Place in incubator for growth – colony develops as
microorganism multiplies

33. Sterile Technique
When culturing bacteria or other
microorganisms, it is important to keep your
work area as clean as possible.
This prevents the introduction of other
microorganisms from the environment into
your culture.
The techniques used to prevent
contamination are referred to as sterile
techniques.
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34. Organise your Work area
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35. Sterile Technique
1. Start by washing
your down your
work or lab benches
with a surface
disinfectant. The
most commonly
used disinfectants
for lab use are:
1. 10% bleach
(recommended by
the CDC)
2. 85% ethanol
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36. Aseptic Technique
• First requirement for study of microbes
–pure cultures, free of other microbes
• Maintain a clean environment; work close to
the flame
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37. Sterile Technique
1. Start by washing your down your
work or lab benches with a surface
disinfectant. The most commonly
used disinfectants for lab use are:
1. 10% bleach (recommended by the
CDC)
2. 85% ethanol
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38. Culturing Specimens
(cont.)
• Culture media
– Liquid, semisolid, or
solid forms
– Contains agar
– Selective or nonselective
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39. Holding the Inoculating loop
11/13/2023 Dr.T.V.Rao MD 39

40. Media Types
• General Purpose Media:
• Supports the growth of many microorganisms
• i.e. Nutrient agar
• Enriched Media:
• Has special nutrients to encourage the growth of fastidious
heterotrophs
• i.e. Blood Agar
• Selective Media:
• Favors the growth of one type of microorganisms and inhibits the
growth of others
• Luria + penicillin Agar
• Differential Media:
• Distinguishes between different groups of bacteria on the basis of
biochemical characteristics
• i.e. Eosin Methylene Blue Agar
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41. Inoculation of Culture Plates and Tubes
 Clean and surface sterilize your work area as detailed
in the section on Sterile Technique.
 Use either disposable inoculation loops or a metal
loop that can be heat sterilized to inoculate plates,
slants, and liquid culture tubes.
If using a metal loop, be sure to cool the loop
by touching the sterile cooled liquid media or
the sterile culture plate before the placing the
loop in your live culture. Failure to cool the
loop will kill your active microbial cultures!
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42. Sterility of the Loop Important in
Culture Work
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43. Inoculating Petri Plates
Step 1:Remove the culture tube stopper or cap with
one (do not set it down) and flame the mouth of
the tube to surface sterilize the mouth. The
heated tube surface will generate a thermal
current that prevents contamination of the
culture.
Step 2: Without setting any of the culture materials
on the bench, place the sterile inoculation loop in
the culture.
Step 3: Replace cap on the culture tube with the
active microbes and put it in the test tube rack.
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44. Culturing Specimens (cont.)
• Inoculating a culture plate
– Transfer some of the specimen onto a culture
plate
– Label the plate correctly
– Qualitative analysis – determination of type of
pathogen
– Quantitative analysis – number of bacteria
present in sample
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45. Inoculating Petri Plates
Step 4: Holding the
petri dish lid at an 30-
45° angle, work the
inoculating loop from
the outside of the
plate toward the
center in a zig-zag
pattern that covers
approximately 25% of
the plate surface .
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46. Inoculating Petri Plates
Step 5: 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 6: 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.
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47. Procedure for Transferring
Microorganisms to a Slant
• 1. Wrap fingers of non dominant hand around the
culture tube containing broth for transfer
• 2. Using the pinkie finger of your dominant hand
twist the red cap from the tube. Hold in your pinkie
and do not place it on the counter
• 3. Pass the mouth of the culture tube across the
flame
• 4. Direct the inoculating needle into the broth.
• 5. Flame the mouth of your broth culture tube and
replace the cap. Place it in your rack
• 6. Pick up the slant in your non dominant hand
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48. Procedure for Transferring
Microorganisms to a Slant
• 7. Twist off the red cap
• 8. Flame the mouth of the slant tube
• 9. Direct the inoculating needle into the tube and “
stab” the agar in the base( butt)
• 10. Withdraw on the entry line and when you reach
the surface make a simple streak along the face.
• 11. Flame the mouth of the tube and replace the
cap.
• 12. Flame your inoculating needle and replace in
your rack.
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49. Culturing Specimens (cont.)
• Inoculating a culture plate
– Transfer some of the specimen onto a culture
plate
– Label the plate correctly
– Qualitative analysis – determination of type of
pathogen
– Quantitative analysis – number of bacteria
present in sample
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50. Triple Streak Method
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51. Optimal results with
Scientific Streaking
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52. Streak plate method of isolation
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53. Colony Morphology
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54. Read Colony Morphology
• Colony
morphology
• Color
• Shape
• Margin
• Elevation
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55. Determining Antimicrobial
Sensitivity
• An outside lab
reports
– Sensitive – no growth
– Intermediate – little
growth
– Resistant –
overgrown
Procedure
Filter paper containing
antimicrobial agents
placed on inoculated
agar plate
Incubated for 24 hours
Evaluate effectiveness of
agent
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56. Microorganism Categories
• How are microorganisms
categorized?
–By genetics to show how they are
related
–By tissues they infect to show how
they cause disease
–By pathogenicity and
communicability (also known as
their Biosafety Level)
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57. Biosafety is a Concern for all
Microbiologists
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58. Biosafety Level 1
Standard Microbiological Practices
• Restrict or limit access
when working
• Prohibit eating, drinking
and smoking in the
laboratory
• Pipetting by mouth
strictly forbidden
2.3
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59. Biosafety Level 1
Standard Microbiological Practices
2.3
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60. Standard practices also include:
• Keep work areas uncluttered and
clean
• No food in lab refrigerator
• Minimize splashes and aerosols
• Decontaminate work surfaces daily
• Maintain insect & rodent control
program
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61. Decontamination
•Sterilization
•Disinfection

62. • Types
–Liquids, i.e.
chlorox,
hydrogen
peroxide
–Gases, i.e.
ethylene oxide
Decontamination
Chemical
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63. • General Lab Use -
Hypochlorite Solutions
–Large Spills/Large Organic Load
• undiluted from bottle
–Small Spills/Virus Inactivation
• 10% - 1:9
–General Surface Disinfection
• 1% - 1:99
Decontamination
Chemical
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64. In case of a spill
• Wear disposable gloves
• Cover large blood spill with paper towels and
soak with 1% (10000 ppm) of household
bleach and allow to stand for at least 5
minutes
• Small spill - wipe with paper towel soaked in
1% bleach
• Discard contaminated towels in infective
waste containers
• Wipe down the area with clean towels soaked
in a same dilution of household bleach
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65. • Programme Created by Dr.T.V.Rao MD
for Medical Microbiologists in the
Developing World
• Email
• doctortvrao@gmail.com
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