4. Aseptic Technique “Pathogen free environment”
Beginning of Lab
1.) Put away Belongings
2.) Clean Tables
3.) wash Hands
4.) Tie back long Hair
BSL-1 (Low risk) Doesn’t cause disease in healthy humans
*Lab Coats
BSL-2 (moderate risk) associated in moderate disease in
humans
*Lab coats & Disposable Gloves
Googles required when using chemicals (Example Gram
staining)
Opportunistic doesn’t typically cause disease in healthy
adults, but may in immunocompromised adults or children
4 potential routes of Exposure:
1. Absorption Through skin or mucous membranes
2. Inhalation
3. Ingestion
4. Inoculation
5. Bacterial Specimen info:
Name of Microorganism
Your name/Initials
Date
Tape labels are required for media in Test Tubes (White)
Carry Test tubes Incubator
Test Tube rack (M/W class Yellow)
Tape petri dishes on both ends before carrying them to
incubator
Disposal of contaminated items:
Petri plates Biohazard waste
Glass pipettes Pipette discard box
Test tubes Test tube discard bin
Glass slides SHARPS box
Gloves Biohazard waste
Plastic ware Biohazard waste
Broken test tubes SHARPS box
Regular Trash uncontaminated items & Paper towels
End of Lab disinfect tables & wash your hands
8. 3 main Prokaryotic Cell shapes
Cocci
Spirals
1. Spirochetes Flexible
2. Spirillum Inflexible
Rods “Bacillis”
Coccobacillus “Short Rod”
Vibrio “Curved Spiral”
Pleomorphic
“Does not Have a shape”
9. Oculars
Arm
Objective
Lenses
Nose Piece
Body
Base
Fine
Focus
Coarse
focus
Stage
Light Source
(Illuminator)
Condenser & Iris
Diaphragm
(underneath
Microscope Skills
Oil Immersion: Apply between 40x and 100x
Specimen in
Field of View & in Focus: Trick Bring the Edge of the slide into
focus should appear as 3-D
Light Level: light source (Illuminator)switchturns light source at base;
Condenser collects& concentrates light from the illuminator& directs
it to the irisdiaphragm
Clean Up: oil Immersioncleaned from all lenses & Stage
Slide removed, Placed in 4x, Stage Lowered & Centered.
Identify: Gram positive/Negative,Morphology, Total Magnification
11. Gram Positive Gram Negative
Teichoic Acid
Lipoteichoic Acid
Peptidoglycan Layer
(cell wall)
Cytoplasmic
Membrane
Integral Protein
Peptidoglycan Layer of
(cell wall)
Cytoplasmic
Membrane
Outer
Membrane of
Cell
Porin Periplasmic
Space
Lipopolysaccharide Layer
(LPS) Containing Lipid A
12. Heat fix
Kills the microbes
Makes microbes more
permeable to stains
Fixes microbes to slide
Why dry before Heat fixing?
If heated right after the water would cause the smear to
overheat & denature some features in the stain.
Bacterial Smear
A smear is a small volume (Loopful) of specimen- containing
medium that is spread (smeared) onto microscope slide
14. Ex. 3 Capsule Stain
Klebsiella Pneumoniae
Capsule is Halo around the
cell
Heat & water
would dislodge Capsule from
bacteria (polysaccharide,
Polypeptide, Glycoprotein) Need t
Air Dry
15. Ex. 4 Acid-Fast Stain
Myobacterium smegmatis (Acid Fast
Cells)
Bacillus Subtilis (Non Acid fast cells)
16. Ex. 5 Endospore Stain(Bacillus & clostridium)
Bacillus Subtilis as Vegatative Cells
Bacillus Subtilis Vegetative cells with Endospore inside
Bacillus Subtilis as a Free Spore
21. Ex. 7 Pure Isolation Cultures
Ilana Kovach (09/02/15) Serratia
Marcescens & Escherichia Coli
21
34
Incubate upside down to prevent
condensation at 25º
Tape both ends
Warning: Sterilize
after each streak
& DO NOT reapply
more bacteria!!!
28. Name of
Specimen in
Broth
Growth Pattern Descriptions
Bacillus Cereus Little Sediment; Mostly Fine
Mycobacterium
Smegmatis
Sediment
Pseudomonas
Aeruginosa
Sediment
Saccromyces
Cerevisiae
Little Sediment; Mostly fine
Serratia
Marscens
Fine
30. Identify the type of Media
in each tube?
1) Broth
2) Slant
3) Deep
Based on the results of this four way isolation
streak, what do you think this student may
have done incorrectly during his or her four
way streak procedure?
They either did not sterilize the loop between
each quadrant or they reapplied the bacteria
after each Quadrant streak
Observe this Circled colony on this agar
plate. Using this provided diagram as a
guide. Determine the following
characteristics?
Size: 5mm
Margin: Entire
Pigmentation: Cream Color
Elevation: Convex
Form: Circular
Observe this Circled colony
on this agar What type of
Growth pattern is shown in
this test Tube
Fine with a little bit of
sediment at the bottom
31. Week 4
Effects of Temperature on Microbial Growth
Oxygen Requirements
Growth of Anaerobic Organisms
32. Ex. 10
Effects of
Temperature on
Microbial Growth
4 nutrient Agar Plates
Split into 5
Incubators:
4◦C, 25◦C, 37◦C & 45◦C
34. Psychrophiles
Cold ~ (-5- 20º); optimal: 10º
Mesophiles
Moderate ~ (20º-45º); optimal: 37º
Thermophiles
Hot ~ (Low 45’s – 80’s)
Hyperthermophiles
HOT!! (80’s & some up to 105º))
Escherichia Coli
Micrococcus Luteus
Serratia Marcescens
Saccharomyces Cerevisiae
Bacillus Stearothermophilus
35. Ex. 11
Oxygen
Requirements
Obtain Melted BHI from
water Bath
While BHI tube is still liquefied add 2 drops of the
appropriate inoculum in (brain Heart infusion)
BHI tube using a sterile Pasteur Pipette; Cap
should be loose (but secure)
Roll BHI tube between palms of your hands to disperse the
bacteria throughout the tube, taking care not to create
bubbles or aerate the agar
Place BHI tube in ice bath until solidify 37ºC incubator
36. Results
Bacillus
Subtilis
Obligate Aerobe
Crinkle at top
(Bacteria
Wants O2)
Saccharomyces
Cerevisiae
Microaerophile
Toward
the Top
Concaved
Specks
toward the
Bottom
Clostridium
Sporogenes
Obligate Anaerobe
Serratia
Marcescens
Film at top
Specks
Throughout
the medium
39. Obligate (Strict)
Aerobes
Requires O2
Undergo Aerobic Respiration
Oxygen finalElectron Acceptor
Microaerophil Require lower O2 levels (2-10%)
Undergo Aerobic Respiration
Limited abilityto detoxifyhydrogen peroxide and superoxideRadicals
Facultative
(Facultative
Anaerobes)
Can Live either Presence OR absence of O2 (Prefer Oxygen)
Aerobic Respiration
Anaerobic Respiration
Fermentation
Aerotolerant Can live in either Presence or Absence of Oxygen (Doesn’t Prefer Oxygen)
Never use Aerobic metabolism
Have enzymes that neutralizetoxicoxygen
Obligate (Strict)
Anaerobes
O2 is Deadly
Use anaerobic Metabolism
Lack Enzymes to neutralizetoxicoxygen
Oxygen Requirements
40. Ex. 12
Growth of Anaerobic
Organisms
Gaspak Jar
The Hydrogen gas combines with free oxygen in the chamber to produce
water. The Reaction is Catalyzed by palladium which is attach to the
underside lid of the Jar . The carbon dioxide replaces the removed
oxygen creating a completely anaerobic environment
2H2 + O2 2H2O
Pseudomonas Aeruginosa
Bacillus Subtilis
Escherichia Coli
Clostridium Sporogenes
Envelope:
Sodium Borohydride H2 (Hydrogen Gas)
Sodium Bicarbonate CO2 (Carbon Dioxide)
41. Results
Candle Jar
Growth Should Occur Under Anaerobic Conditions:
Facultative Escherichia Coli
Obligate Anaerobe Clostridium Sporogenes
Clostridium Sporogenes “Obligate Anaerobe”
Escherichia Coli “Facultative”
43. Ex. 12
Growth of Anaerobic
Organisms
4 Thioglycollate Broth Tubes
Pseudomonas Aeruginosa
Bacillus Subtilis
Escherichia Coli
Clostridium Sporogenes
Pink = Oxygen
Yellow = No Oxygen
Resazurin The oxygen concentration at a given level is
indicated by a redox sensitive dye that turns pink in the presence of
oxygen
Sodium Thioglycollate Consumes Oxygen
45. Identify the Oxygen Class & any Example
Genus +Species
1) Aerotolerant Enterococcus Faecalis
2) Obligate Aerobe Bacillus Subtilis
3) Microaerophil Saccharomyces Cerevisiae
4) Facultative Escherichia Coli
5) Obligate Anaerobe Clostridium Sporogenes
Indicate Temperature Class Name the Chemical that is the
reducing agent in the Media?
Sodium Thioglycollate
Name the chemical in this media
that changes color depending on
whether or not oxygen is present
Resazurin
Oxygen Class Tube A Facultative
Oxygen Class Tube B Aerotolerant
Observe this Circled colony Name 2
chemicals in Gas Pak Envelope
1) Sodium Bicarbonate
2) Sodium Borohydride
2 chemicals Produced when chemicals in
Envelope are Activated
CO2 & O2
Write Equation that occurs in GASPAK
Jar that removes O2 and makes chamber
Anaerobic
2H2 + O2 2H2O (Pallidium Catalyst)
4◦C 25◦C 37◦C 55◦C Temperature Class
A - + - - Mesophile
B + + - - Psychrotroph
C - - - + Thermophile
D - + + - Mesophile
E + - - - Psychrophile
46. Week 5
Selective & Differential media
Chemical control of Microorganisms
Chemotherapeutic agents
Additive & synergistic effects on Antibiotics
47. Ex. 13: Use of
Selective, Differential &
Enriched Media
Mannitol Salt Agar
Selective: 7.5% NaCl
Halotolerants
Differential: Carbohydrate Substrate Mannitol &
“phenol Red” (Ability to ferment mannitol)
Red Yellow
fermentation
No fermentation
Staphylococcus xylosus
Enterococcus faecalis
Staphylococcus epidermidis
48. Blood Agar
Differential ONLY: Hemolysis some bacteria produce exotoxins that cause lysis of RBC
Beta (Burst): Complete Clearing of Blood around colonies
Alpha (almost): Partial lysis Greenish-Yellow Discoloration
Gamma: No lysis
Streptococcus mutans
GammaAlphaBeta
Enterococcus faecalis
Staphylococcus Xylosus
Streptococcus Zooepidmicus
49. Columbia
C-NA agar
Selective: Colistin + Nalidixic Acid
Gram Positive
Differential: Blood agar
Beta, Alpha, Gamma
Alpha
Enterococcus Faecalis
Staphylococcus xylosus
Beta
50. MacConkey
Agar
Selective: Crystal Violet & Bile Salts
Gram Negative
Differential: Substrate Lactose & pH
indicator “Neutral Red”
RED Pink
Escherichia Coli
Enterobacter aerogenes
Morganella Morganii
51. Eosin Methylene Blue Agar
Selective: Eosin + Methylene Blue
Gram Negative
Differential: Substrate Lactose
Pink Small amount of acid
Greenish Large amount of acid
Escherichia Coli
Enterobacter aerogenes
Morganella Morganii
52. Ex. 14:
Chemical Control of
Microorganisms
Measuring Zone of Inhibition
Bleach
Lab Cleanerisopropanol
Vinegar
53. Ex. 15:
Chemotherapeutic
Agents
E test
Antibiotic: Trimethoprim & sulfa TS
MIC .047
Susceptible
S < R >
Ampicillin (AM) 8 8
Daptomycin (DPC) 1 1
Imipenem (IP) 2 8
Tetracycline (TC) 1 2
Trigecycline (TGC) 1 2
Trimethoprim & Sulfa
(TS)
2 4
56. Ex. 16: Additive & Synergistic Effects on Antibiotics
Synergistic
Additive
“Overall no
greater than the
sum of individual
antibiotic.
Uniform”
“Overall effect of
antibiotic combination is
greater than sum of
individual antibiotic.
Merge in the middle”
57. Station 3: MSA plates
1) What does the MSA select for? Halotolerants
2) What are the selective ingredients of MSA? 7.5% sodium chloride
3) What is the carbohydrate in MSA? Mannitol
4) Does the bacteria growing in sector A ferment mannitol? YES
5) Does the bacteria growing on sector B ferment mannitol? NO
6) Give example of a bacterium that ferments mannitol and one that does not ferment mannitol
a. Enterococcus Faecalis
b. Staphylococcus Epidermis
Station 1: EMB and MacConkey Plate
1) What does EMB and MacConkey Plates Select for? Gram –
2) What carbohydrates is in both EMB and MacConkey agar? Lactose
3) What are the selective ingredients in EMB agar? Eosin & Methylene Blue
4) What are the selective ingredients in MacConkey agar?
Crystal Violet & Bile salts
1) What is the pH indicator in MacConkey agar? Neutral Red
2) Does the bacteria growing on sector A ferment Lactose?
EMB (No) & MacConkey (No)
1) Does the bacteria growing on sector B ferment Lactose?
EMB (Yes) & MacConkey Yes
1) Give an example of a bacteria that ferments lactose
and one that does not ferment lactose
a. Escherichia Coli
b. Morganella Morganii
58. Station 3: Mueller-Hinton Plates
1) Measure the zone of Inhibition SXT25 on the disk fusion plate? 23mm
2) Based on your measurement, is the bacteria on this plate susceptible,
resistant or intermediate to this antibiotic. Susceptible
3) Measure the MIC at .032 on the E-test plate. .032
Station 4: CCNA plate
1) What does CCNA select for? Gram -
2) Name the two antibiotics that make CCNA agar selective Colistin & Nalidixic Acid
3) Identify the hemolysis pattern of the bacteria growing on sector A “alpha”
4) Identify the hemolysis pattern of the bacteria growing on sector B “Beta”
5) Give an example of (a) bacterium that is alpha hemolytic (b) Beta Hemolytic (c)
Gamma hemolytic
a. Enterococcus Faecalis (alpha)
b. Staphylococcus xylosus (Beta)
c. Streptococcus Mutans
59. Week 6
Normal microbiota of the mouth
Normal microbiota of the throat & Skin
Cultivation of Urine Specimen
60. Ex. 17: Microbiota of the Mouth
Unbrushed teeth is an accumulation of a biofilm
Cariogenic- Ability to cause dental Carries
*lactobacillus acidophilus, streptococcus mutans (S. Gordonii, S.
salivarius) & actinomyces odontolyticus
Ferment carbohydrates Lactic Acid
Lowering the pH at the surface of the Enamel which can
decalcify the enamel & lead to cavities
S. Mutans produce enzyme Dextransucrase which
polymerizes sucrose to form a polysaccharide called Glucan
L. Acidophilus produces similar polymer Lexan
Glucan & Lexan are both Glycocalyces
61. Snyder Test
Determine a Persons Susceptibility to dental caries
based on acid production that is assumed to be a result of the
growth of lactobacilli
Ingredients Select: 2% glucose & pH of 4.8
Low pH inhibits growth of most organisms but is ideal for lactobacilli
Indicator Differential: Bromcresol Green (4.84.4)
* Green Yellow
Green = Slight
Yellow = High
Slight mild Moderate High
Fermenting to Yellow
62. Ex. 18: Microbiota of the Throat & Skin
Blood Agar
Differentiate: Streptococcus
S. Pyogens Beta Hemolysis (Burst)
S. mutans, S. gordanii, S. salivarius Alpha Hemolysis (almost)
Chocolate Blood Agar
NOT selective
“lysed Boiled Red blood Cells”
Allows growth of Neisseria & Moraxella
Oxidase Reagent Detects (Cytochrome C) Turn Purple 30s
Common in young Adults as normal microbiota
Neisseria &
Moraxella
Oxidase Reagent Detects
(Cytochrome C)
Streptococcus
Pyogens
Beta
S. mutans
S. gordanii
S. salivarius
Alpha
63. MSA Plate
Select: Staphylococcus/ Halotolerant “7.5% NaCl”
Differentiate: Mannitol Ferment “pH indicator Phenol Red”
Staphylococcus Epidermidis (drier) Does not Ferment
Staphylococcus Aureus (moist) Ferments
M-H Tellurite Plate
Select: Corynebacterium Diptheroids
Present produce gray/Black colonies
Tellurite in the media is reduced intracellularly Tellurium (grey precipitate)
“Back of ear”“arm… used 0.85% saline swab”
Corynebacterium
Diptheroids
Staphylococcus
Aureus
Staphylococcus
Epidermidis
64. Environmental
Organisms
Sabouraud agar
Fungi (yeast or Mold)
Selects: Acidophiles (acidic Medium pH5.5)
Nutrient Agar Plate
Not Selective or Differential Acidophils will not grow
Acidophil grows
(Fungi)
65. UTI’s
Urethritis: Infection of the Urethra
Cystitis: Infection of the urinary bladder
Pyelonephritis: Infection involving kidneys
* Caused by … E. Coli, Staphylococcus Aureus,
Enterococcus Faecalis, Proteus mirabilis & P. Vulgaris
Micturition in to a Cup Midstream *adequate cleaning
of the genitals
HardyCHROM is a Non-selective/ differential
Ingredients: Peptones, Proprietary blend of chromogenic
substrates & indicators that cause different organisms to
produce different colored colonies
# 𝒐𝒇 𝒄𝒐𝒍𝒐𝒏𝒊𝒆𝒔
𝟎.𝟎𝟏𝒎𝒍
=
𝑪𝑭𝑼
𝒎𝒍
Critical threshold = 1*105 bacteria per ml of urine
Early phase of UTI = 1*102 to 1*104
Ex. 19:Cultivation of Urine Specimen
67. Station 2: Skin Microbiota
1) What genus of the skin microbiota does the MSA select for? Staphylococcus
2) What species of the skin microbiota is growing on sector A? Aureus
3) What species of the skin microbiota is growing on sector B? Epidermidis
4) Give the common name & genus of the bacteria growing on the MHT plate Diphtheria
colonies, Corynebacterium
5) What is the name of the metal in this plate that selects for the bacteria on MHT? Tellurite
Station 1: Microbiota of the Mouth
1) What is the name of the agar we use to test for susceptibility to dental caries? Snyder deep agar
2) What genus of bacteria does this agar test for? Lactobacillus, Streptococcus & acintomyces
3) What makes this agar selective? The pH from 2% glucose
4) What carbohydrate is in this agar? Glucose
5) What is the name of the ph indicator in this agar? Bromersol Green
6) what causes the color of the agar to change from green to yellow? Color change is caused by acid
production
Station 3: Throat Microbiota
1) Name the species of throat microbiota that is growing on this blood plate? Pyogenes (Beta Hemolytic)
2) What is the main ingredient in chocolate agar? Lysed boiled red blood cells
3) What genus of throat microbiota doe we culture on chocolate agar? Neisseria & Moraxella
4) What reagent has to be added to the growth on the plate to detect this genus? Oxidase Reagent
68. Station 4: Urine Cultures
1) Name the pathogen growing on sector A of the HardyCHROM plate? Escherichia Coli
2) Name the pathogen growing on Sector B of the HardyCHROM plate? Cirobacter
3) 0.01ml of a urine specimen is streaked onto a nutrient agar plate, and after incubating the plate
overnight at 3 7 degrees C. 263 colonies are present on the plate. Calculate how many bacteria
were in the patients urine sample. Does the value mean that the patient has a UTI?
263
.01
= 26,300 2.63 * 104 NO UTI less than 105
Snyder: Selective & Differential
MSA: Selective & Differential
MHT: Selective
Blood: Differential
Chocolate: Differential
Sabouraud: Selective
Nutrient: Neither
E. Coli
Rose/Magenta
Cirobacter
Dark Blue With pink Halo