Culture Flashcards
(300 cards)
1
Q
PRINCIPLES OF BACTERIAL CULTIVATION
A
✓Grow and isolate all bacteria present in a
clinical specimen
✓Determine which of the bacteria that grow
are most likely causing infection and which
are likely contaminants
✓Obtain sufficient growth of clinically relevant
bacteria to allow identification,
characterization, and susceptibility testing
2
Q
process of growing microorganisms in culture by taking bacteria
from the infection site (in vivo environment) by some means of
specimen collection and growing them in the artificial
environment of the laboratory (in vitro environment)
A
Cultivation
3
Q
→nutrient material prepared for the growth of microorganisms in a
laboratory
A
Culture Medium
4
Q
composed of mixture of nutrients: Carbon, Nitrogen, Sulfur,
Phosphorus, Hydrogen, Oxygen and Buffer
A
Culture Medium
5
Q
Inhibitory agents→ facilitate isolation of desired organism while
suppressing the growth of other organism
A
Culture Medium
6
Q
microbes that are introduced into a culture medium to initiate
growth
A
Inoculum
7
Q
microbes that grow and multiply in or on a culture
medium
A
CULTURE
8
Q
Types of CULTURE
A
Pure (Axenic) Culture
Mixed Culture
Stock Culture
9
Q
Composed of only one species
A
Pure (Axenic) Culture
10
Q
Composed of more than one species
A
Mixed Culture
11
Q
Composed of several species contained in a separate culture medium—one specie per culture medium
A
Stock Culture
12
Q
Grown in a large volume of broth and then divided into small freezer vials—lengthen the shelf life of specimen to at least a year
A
Stock Culture
13
Q
Sulfated polymer made up of D-galactose, 3,6-anhydro-Lgalactose, and D-glucoronic acid and usually derived from red
algae
A
Agar
14
Q
melt at 80°C-90°C (100°C) and solidify at 40°C-50°C
A
Agar
15
Q
cooling temperature for distribution of culture
medium into Petri plates
A
55°C-60°C
16
Q
Amount of molten agar transferred to a sterile
plates
A
20-25 ml
17
Q
CLASSIFICATION OF CULTURE MEDIA
A
I. According to Physical State or Consistency
II. According to Composition
III. According to the Dispensing or Distribution
Method for the Medium
IV. According to Use
18
Q
(CULTURE MEDIA)
According to Physical State or Consistency
A
Liquid Medium
Semi-solid Medium
Solid Medium
19
Q
does not contain any amount of agar or solidifying
substances
A
Liquid Medium
20
Q
allows growth of aerobes, anaerobes and facultative
anaerobes
A
Liquid Medium
21
Q
Example of Liquid Medium
A
✓Nutrient Broth
✓Brain Heart Infusion (BHI)
✓Trypticase Soy Broth (TSB)
✓Thioglycollate (THIO)
22
Q
contains 0.5% to 1% agar
A
Semi-solid Medium
23
Q
observed bacterial motility and detect indole and
sulfide production
A
Semi-solid Medium
24
Q
Example of Semi-Solid Medium
A
Sulfide Indole Motility (SIM) Medium
25
contains 2% to 3% agar
Solid Medium
26
(CULTURE MEDIA)
According to Composition
Synthetic or Defined Medium
Non-synthetic or Complex Medium
Tissue Culture Medium
27
exact chemical composition of the ingredients
is known (commercially prepared culture media)
Synthetic or Defined Medium
28
used for research purposes as either a liquid or
solid medium
Synthetic or Defined Medium
29
preferred for the isolation of cyanobacteria and
chemoorganotrophs
Synthetic or Defined Medium
30
Example of Synthetic or Defined Medium
BG-11 medium
31
precise composition of some or all of the nutritive
substances used is not known (Peptone, Meat and Yeast Extracts)
Non-synthetic or Complex Medium
32
useful for the isolation of medically significant bacteria
Non-synthetic or Complex Medium
33
Example of Non-synthetic or Complex Medium
Nutrient Broth (NB) Medium, TSB and MAC Agar
34
contains living tissues
Tissue Culture Medium
35
used for obligate intracellular bacteria---Rickettsia and Chlamydia
Tissue Culture Medium
36
Example of Tissue Culture Medium
HeLa 229→ human cervical tissue, Chlamydia
McCoy and W 138→ fibroblasts, Chlamydia
Embryonated Egg→ propagation of Rickettsia
37
(CULTURE MEDIA)
According to the Dispensing or Distribution Method for the Medium
Plated Media
Tubed Media
38
distributed into sterile petri dish
Plated Media
39
distributed in sterile test tube
Tubed Media
40
Types of TUBED MEDIA
1. Slant
2. Butt-Slant
3. But
41
Example of Tubed Media
TSI, SIM, Simmon’s Citrate Agar (SCA),
Lysine Iron Agar (LIA)
42
(CULTURE MEDIA)
According to Use
1) Simple Media, Supportive Media or General Purpose Media
2) Enrichment Media
3) Enriched Media
4) Differential Media
5) Selective Media
43
routinely used in the laboratory and without additional supplement
Simple Media, Supportive Media or General Purpose Media
44
support growth of most non-fastidious bacteria to grow at natural rates, without providing advantage to any particular bacteria
Simple Media, Supportive Media or General Purpose Media
45
usually composed of meat and soybean extracts
Simple Media, Supportive Media or General Purpose Media
46
Example if Simple Media, Supportive Media or General Purpose
Media
✓Nutrient Agar
✓Nutrient Broth
✓TSB
47
enhance the growth of particular organisms (pathogens) and
suppress the growth of normal flora present in specimen
Enrichment Media
48
contain specific nutrients and without additional supplements
Enrichment Media
49
incubated for a certain period and then subcultured to
isolate the desired organism
Enrichment Media
50
can also be used as a supplement to agar plates to detect
aerobes, anaerobes and microaerophlies (THIOGLYCOLLATE)
Enrichment Media
51
Example of Enrichment Media
✓Alkaline Peptone Water (APW)
✓Selenite F
✓Thioglycollate
✓Tetrathionate
✓Gram-Negative Broth
✓Lim Broth (Todd Hewitt with CNA)
52
→promote growth of Vibrio spp. before inoculation
into Thiosulfate-Citrate-Bile-Salts(TCBS) Agar
→adjusted to pH 8.5
Alkaline Peptone Water (APW)
53
isolation of Salmonella from feces, urine and water sample
Selenite F
54
general support enrichment medium that promotes the
growth of almost all non-fastidious bacteria
Thioglycollate
55
Components of Thioglycollate:
▪ Dextrose, Vitamin K1, and Hemin have been used to
modify the basic thioglycollate formula
▪ 0.075% agar
▪ Resazurin→ oxidation-reduction indicator
▪ Thioglycolic Acid→ reducing agent
56
-Selective enrichment broth for the isolation of
Salmonella and Proteus
-Bile Salt and Thiosulfate→ suppresses the growth of
other coliform bacilli
Tetrathionate
57
Bile Salt and Thiosulfate
suppresses the growth of
other coliform bacilli
58
solation of Salmonella and Shigella
Gram-Negative Broth
59
Enrichment and Selective medium
Gram-Negative Broth
60
→ inhibit gram-positive organisms
Sodium Citrate and Sodium Desoxycholate (a
bile salt)
61
→ primary carbon source
Mannitol
62
Gram-Negative Broth
Sodium Citrate and Sodium Desoxycholate (a
bile salt)
Mannitol
63
Group B Streptococci
Lim Broth (Todd Hewitt with CNA)
64
→media with additional supplements necessary
for growth of fastidious organisms
Enriched Media
65
Supplements: Blood, Vitamins, Serum, Peptone
and Yeast Extract
Enriched Media
66
solid type media
Enriched Media
67
Example of Enriched Media
✓Blood Agar Plate (BAP)
68
contains 5% defibrinated blood
Blood Agar Plate (BAP)
69
Differentiate haemolytic pattern of
bacteria
Blood Agar Plate (BAP)
70
Choices of blood in Blood Agar Plate
Sheep, Horse, Rabbit
71
→blood has been chemically-treated or heat-treated
(80°C) to lyse the RBC
✓Chocolate Agar Plate
72
isolation of fastidious microorganisms:
***Neisseria gonorrhoeae and Haemophilus spp.
✓Chocolate Agar Plate
73
(Chocolate Agar Plate)
isolation of fastidious microorganisms:
_______
Neisseria gonorrhoeae and Haemophilus spp.
74
“X” factor
Hemin
75
“V” factor
Nicotinamide Adenine Dinucleotide (NAD)
76
allow the visualization of metabolic differences
between groups of bacteria
Differential Media
77
distinguishes organisms growing together by their
diffrences in cultural characeristics
Differential Media
78
allow grouping of microbes based on different
characteristics demonstrated on the medium
Differential Media
79
Example of Differential Media
BAP
Eosin Methylene Blue (EMB)
Hektoen Enteric Agar (HEA)
80
→Lactose and Sucrose
→Eosin and Methylene Blue
Eosin Methylene Blue (EMB)
81
→differentiate Lactose Fermenter (pink colonies)
from Non-Lactose Fermenter (colorless colonies)
MacConkey Agar
82
Components of MacConkey Agar
▪ Lactose
▪ Bile Salts
▪ Crystal Violet→ inhibit gram-positive bacteria and
fungi
▪ Neutral Red→ pH indicator
83
(MacConkey Medium)
pH indicator
Neutral Red
84
(MacConkey Agar)
inhibit gram-positive bacteria and
fungi
Crystal Violet
85
support the growth of one type or group of microbes but not
another
Selective Media
86
contain inhibitory substances such as antimicrobials, dyes, or
alcohol which inhibit the growth of other organisms while
promoting the growth of the desired organism
Selective Media
87
INHIBITORY AGENTS
1) Inhibit growth of Gram-Positive Microorganism
2)Inhibit growth of Gram-Negative Microorganisms
3) Prevent Swarming of Proteus
88
Inhibit growth of Gram-Positive Microorganism
✓Crystal or Gentian Violet
✓Basic or Carbol Fuchsin
✓Bile Salts
✓Sodium Desoxycholate
89
Inhibit growth of Gram-Negative Microorganisms
✓Potassium Tellurite
✓Sodium Azide
90
Prevent Swarming of Proteus
✓Alcohol
✓Chloral Hydrate
91
Examples of Selective Media
✓HEA
✓MAC
✓Xylose Lysine Deoxycholate (XLD)
✓Bismuth Sulfite Agar (BSA)
✓Mannitol Salt Agar (MSA)
✓Thayer Martin Agar (TMA)
✓Salmonella-Shigella Agar (SSA)
✓TCBS
92
→ Salmonella spp. and Shigella spp.
→bile salts and dyes (bromthymol
blue and acid fuchsin)
Hektoen Enteric (HE) Agar
93
Hektoen Enteric (HE) Agar
pH indicator
Bromthymol Blue
94
(Hektoen Enteric (HE) Agar)
→ H2S indicator
Salmonella→ black precipitate
Ferric Ammonium Citrate
95
Shigella spp. and Salmonella spp.
Xylose-Lysine-Desoxycholate (XLD) Agar
96
Components Xylose-Lysine-Desoxycholate (XLD) Agar
▪Lysine, Lactose, Xylose and Sucrose
▪0.25% Sodium Desoxycholate
→ inhibits gram-positive
bacteria
▪Phenol Red→ pH indicator
Ferric Ammonium Citrate→ H2S indicator
97
(Xylose-Lysine-Desoxycholate (XLD) Agar)
inhibits gram-positive
bacteria
▪0.25% Sodium Desoxycholate
98
(Xylose-Lysine-Desoxycholate (XLD) Agar)
pH indicator
Phenol Red
99
(Xylose-Lysine-Desoxycholate (XLD) Agar)
H2S indicator
Ferric Ammonium Citrate
100
colonies are red with
black center
Salmonella
101
MEDIA FOR GRAM-POSITIVE BACTERIA
1) Columbia CNA with Blood
2) Phenylethyl Alcohol (PEA) Agar
102
three peptone sources and 5% defibrinated
sheep blood
Columbia CNA with Blood
103
suppress the growth of most gram-negative
organisms
Colistin (C) and Nalidixic Acid (NA)
104
sheep blood agar supplemented with
phenylethyl alcohol to inhibit the growth of
gram-negative bacteria
Phenylethyl Alcohol (PEA) Agar
105
Culture Media for Neisseria spp.
1) THAYER-MARTIN
2) MODIFIED THAYER-MARTIN AGAR
3) TRANGROW MEDIUM
4) MARTIN-LEWIS AGAR
5) NEW YORK CITY MEDIUM
106
enriched Chocolate Agar with supplement B or
Isovitale X
THAYER-MARTIN
107
Antibiotic Components:
▪ Colistin
▪ Vancomycin
▪ Nystatin
108
(MODIFIED THAYER-MARTIN AGAR & THAYER-MARTIN)
Antibiotic Component that
inhibit gram-negative bacteria
Colistin
109
(MODIFIED THAYER-MARTIN AGAR & THAYER-MARTIN)
Antibiotic Component that inhibit gram-positive bacteria
Vancomycin
110
(MODIFIED THAYER-MARTIN AGAR & THAYER-MARTIN)
Antibiotic Component that inhibit yeast
Nystatin
111
→Neisseria gonorrhoeae and Neisseria meningitides
→chocolatized blood + antibiotics
MODIFIED THAYER-MARTIN AGAR
112
Antibiotic components
▪ Colistin
▪ Vancomycin
▪ Nystatin
▪ Trimethoprim Lactate
113
(MODIFIED THAYER-MARTIN AGAR)
Antibiotic component that inhibit Proteus spp
Trimethoprim Lactate
114
Thayer-Martin with glucose, 2% agar,
Trimethoprim Lactate and CO2
incorporated in bottle
TRANGROW MEDIUM
115
substitute Anisomycin*** for Nystatin and
higher concentration of vancomycin
MARTIN-LEWIS AGAR
116
Modified Thayer-Martin with substitution of
Amphotericin B*** for Nystatin
NEW YORK CITY MEDIUM
117
OTHER SELECTIVE MEDIA
Gentamicin Blood Agar
Bacitracin Chocolate Agar
Blood Agar Plate with Ampicillin
118
Gentamicin Blood Agar
Streptococcus
119
Bacitracin Chocolate Agar
Haemophilus
120
Blood Agar Plate with Ampicillin
Aeromonas
121
used in the primary isolation of enteric Gram-Negative bacteria
Selective and Differential Media
122
Example of Selective and Differential Media
✓Endo Agar
✓XLD Agar
✓MAC Agar
✓EMB Agar
123
solate bacteria with specific growth requirements
Special Media
124
specially prepared to support the growth of specific
microorganisms
Special Media
125
Examples of Special Media
✓Middlebrook 7H-10 Agar
✓Fletcher Medium
✓“W”or Winsconsin Medium
✓Bordet-Gengou Agar
✓Thayer Martin
✓MacBride
✓Dieudonn’s Medium
126
Middlebrook 7H-10 Agar
M. tuberculosis
127
Fletcher Medium
Leptospira
128
“W”or Winsconsin Medium
Brucella
129
Bordet-Gengou Agar
Bordetella pertussis
130
Thayer Martin
Nesseria
131
Dieudonn’s Medium
Vibrio cholerae
131
MacBride
Listeria monocytogenes
132
protein rich medium composed of whole
egg and malachite green and supports
the growth of Mycobacteria
Lowenstein-Jensen
133
Sterilization: Inspissation not Autoclaving
Lowenstein-Jensen
134
selective for the isolation of Vibrio
Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS)
Agar
135
“Special Medium"
Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS)
Agar
136
Sterilization: Boiling not Autoclaving
Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS)
Agar
137
General Steps in Preparation of Culture Medium
TUBE METHOD
PLATED MEDIUM
INOCULATION OF MEDIA
138
TUBE METHOD
1. Weighing
2. Dissolving
3. Titration
4. Distribution
5. Sterilization
139
→ weigh the different ingredients then
place in clean, dry containers
Weighing
140
→ add the exact amount of solvent to
the ingredients and then dissolve by
heating
Dissolving
141
→ adjustment to the right pH : 7.2-7.4
Titration
142
→ distribute in test tubes
Distribution
143
PLATED MEDIUM
1.Weighing
2.Dissolving
3.Titration
4.Sterilization
5.Distribution
144
INOCULATION OF MEDIA
SPECIMEN CONSIDERATIONS
1. Sterile body fluids, pus, urine and sputum
2. Specimens received on swabs
3. Specimens that require direct or “bedside”
inoculations
145
Inoculated directly into the selected media
Sterile body fluids, pus, urine and sputum
146
Can be inoculated directly into the culture
media
Specimens received on swabs
147
Specimens that require direct or “bedside”
inoculations
Blood, Genital specimens, Corneal
scrapings, Sterile fluids like Synovial and
Peritoneal fluids and Nasopharyngeal Swabs for
isolation of Bordetella pertussis
148
most common manner of inoculation
Streaking
149
Placement of fluid specimen or swabs into a broth or
liquid culture media
Streaking
150
✓Stabbing of medium is usually performed with group A _______________ to create anaerobiosis and promote sub-surface hemolysis
streptococci
151
inoculation technique used for antimicrobial sensitivity test
Overlapping inoculation
152
Inoculation of Tubed Media
a. Liquid Medium
b. Slant Medium
c. Butt Medium
d. Butt/Slant Medium
153
Liquid medium is inoculated with the use of
Sterile Pasteur Pipet
154
➢inoculate by shaking a previously heated wire loop or needle
Liquid Medium
155
➢With the use of a wire loop or needle, transfer the inoculum to the bottom of the slant and streak in a zig-zag manner across the entire surface toward the mouth of the tube
Slant Medium
156
➢Just stab the medium with an inoculating
needle
Butt Medium
157
➢Inoculate the butt first by stabbing the
needle to the bottom of the medium and
then streak the surface in a zig-zag manner
toward the mouth of the tube
Butt/Slant Medium
158
Inoculation of Plated Media
a. Streak Plate Technique
b. Pour Plate
c. Streak-Pour Plate
159
inoculate by isolating organisms in pure culture
Streak Plate Technique
160
Inoculation that determine the approximate number of viable organisms in a liquid such as water, milk, urine or broth culture
Pour Plate
161
Inoculation for studying hemolysis
Streak-Pour Plate
162
→calibrated loop inserted into the
urine and transferred to the culture
medium by making a single streak
down the center of the plate
→without flaming, the loop is
streaked back and forth through
the original inoculum
Quantitative Isolation
163
What is the specimen used for Quantitative Isolation?
Urine Specimens
164
Quantitative Isolation use a calibrated loop to deliver a specified volume of
0.01 or 0.001 mL
165
Manner of Reporting (Grading) of Growth on Plate
4+
many, heavy growth; growth is up to
the fourth quadrant
166
Manner of Reporting (Grading) of Growth on Plate
3+
moderate growth; growth is up to
the third quadrant
167
Manner of Reporting (Grading) of Growth on Plate
2+
few or light growth; growth is in the
second quadrant
168
Manner of Reporting (Grading) of Growth on Plate
1+
rare growth; growth is in the first
quadrant only
169
METHODS OF OBTAINING PURE CULTURE
✓Streak-plate method
✓Pour- plate method
✓Use of selective media and media
containing antibiotic
✓Animal inoculation test
170
→usually measured in millimeters or described as pinpoint, small, medium, large
Colony Size
171
→includes form, elevation, and margin of the colony
Colony Shape
172
→glistening, opaque, dull, dry, transparent
Colony surface appearance
173
→hemolytic pattern on blood agar, changes in color of pH indicators, pitting of the agar surface
Changes in agar media resulting from bacterial growth
174
→certain bacteria produce distinct odors that can be helpful in preliminary identification
Odor
175
COLONY MORPHOLOGY
✓Colony size
✓Colony pigmentation
✓Colony shape
✓Colony surface appearance
✓Changes in agar media resulting from bacterial growth
✓Odor
176
Types of Colony
A. Mucoid (M) Colony
B. Smooth (S) Colony
C. Rough (R) Colony
177
(Colony)
→exhibits a water-like, glistening, confluent appearance
→characteristic of organisms that form slimes or welldeveloped capsule
Mucoid Colony
178
(Colony)
→uniform texture and homogeneity
→asily emulsified in NSS
→haracteristic of freshly isolated wild type
microorganisms (virulent microorganisms)
Smooth Colony
179
Examples of Bacteria in M Colony
K. pneumoniae, S. pneumoniae, H. influenzae
180
Examples of Bacteria in S Colony
Salmonella, Shigella, E. coli, Serratia, Proteus
181
(Colony)
→granulated and rough in appearance
→hard to emulsify in NSS
→usually produced by mutant strains that lack
surface proteins or polysaccharides indicating loss of virulence
Rough (R) Colony
182
Examples of Bacteria in R Colony
Rough forms of enteric bacteria
183
Exception for Rough Colony
R forms of B. anthracis and human and
bovine types of M. tuberculosis (more
virulent)
184
(Colony Characteristic)
→observed in the media immediately surrounding or underneath the colony is a reaction caused by enzymatic or toxin activity of bacteria
→presumptive identification of streptococci and enterococci
Hemolysis
185
→partial lysing of erythrocytes in a BAP around and under the colony that results in a green discoloration of the medium
α-Hemolysis
186
Examples of Bactria for α-Hemolysis
Streptococcus pneumoniae and certain
viridans streptococci
187
Culture Media for Hemolysis
Blood Agar Plate
188
→complete clearing of erythrocytes in BAP around or under the colonies because of the complete lysis of RBCs
β-Hemolysis
189
wide, deep, clear zone
of β-hemolysis,
Streptococcus pyogenes
190
→narrow, diffuse zone of β-hemolysis close to the colony
Streptococcus agalactiae and Listeria
monocytogenes
191
(Colony Characteristic)
→ described as large, medium, small, or pinpoint
→gram-positive bacteria produce smaller
colonies than gram-negative bacteria
Size
192
In BAP, small, white colonies are
gram-positive cocci
193
In BAP, large, gray, mucoid colonies are
enteric gram-negative rods
194
(Colony Characteristic)
→edge of the colonies
→described as smooth, filamentous, rough or rhizoid, or irregular
Form of Margin
195
“Medusa Heads”---filamentous appearance
Bacillus anthracis
196
swarming phenomena in what bacteria
Proteus mirabilis and Proteus vulgaris
197
hazy blanket of growth on the
surface that extends well beyond
the streak lines
Swarming
198
bacteria with rough edges
Diphtheroids
199
(Colony Characteristics)
→determined by tilting the culture
plate and looking at the side of the
colony
→raised, convex, flat, umbilicate
(depressed center, concave—an
“innie”), or umbonate (raised or
bulging center, convex—an “outie”)
Elevation
200
umbilicate colonies (unless the colonies
are mucoid)
S. pneumoniae
201
→ convex colonies β-hemolytic streptococci
→ flat colonies
S. aureus
202
(Colony Characteristics)
→transparent, translucent, or opaque
Density
203
Density of β-Hemolytic streptococci except group B (S. agalactiae)
→ translucent
204
Density of S. agalactiae
semiopaque
205
Density of Staphylococci and other gram-positive bacteria
Opaque
206
Density of most gram-negative rods
opaque
207
Density of Bordetella pertussis
→ shiny, similar to a half-pearl,
on blood-containing media
208
Color of Coagulase-negative staphylococci
white
209
Color of Enterococcus spp.
gray
210
Color of Certain Micrococcus spp. and Neisseria (nonpathogenic) spp.
yellow or off-white
211
Color of Diphtheroids
buff
212
Color of most gram-negative rods
gray on BAP
213
(Colony Characteristic)
→determined by touching the colony with a sterile loop
→ brittle (splinters), creamy (butyrous), dry, or waxy
Consistency
214
Consistency of S, aureus
Creamy
215
COnsistency of Nocardia spp.
brittle, crumbly, and wrinkled, resembling bread crumbs on a plate
216
Consistency of diphtheroid
dry and waxy
217
Pseudomonas aeruginosa pigment
green
218
Serratia marcescens pigment
brick-red (especially at room temperature)
219
Chromobacterium violaceum pigment
purple
220
Prevotella meninogenica pigment
brown-black (anaerobic)
221
Kluyvera spp. pigment
blue
222
Odor of S. aureus
old sock---Mannitol Salt Agar
223
Odor of P. aeruginosa
fruity or gapelike
224
Odor of P. mirabilis
putrid
225
Odor of Haemophilus spp.
musty basement, “mousy” or
“mouse nest” smell
226
Nocardia spp. odor
freshly plowed field
227
OTHER MEDIA for Staphylococcus aureus
✓CNA
✓Chapman stone agar
✓Vogel-Johnson medium
✓PEA, MSA
228
OTHER MEDIA for Streptococci
✓ PEA, Todd-Hewitt broth, CAN
229
OTHER MEDIA for Neisseria
✓ Thayer-Martin
✓ Modified Thayer-Martin
✓ Transgrow medium
✓ Martin-Lewis
✓ New York City medium
230
OTHER MEDIA for Mycobacterium
✓ Dubos oleic acid medium
✓ Middlebrook 7H10 or 7H11
✓ Mitchison’s selective 7H11
✓ Petragnani
✓ LJ
✓ Dorset egg
✓ American Thoracic Society
medium
✓ Bactec 12B medium
✓ Middlebrook 7H9 broth
231
OTHER MEDIA for Corynebacterium diphthriae
✓Loeffler’s coagulated serum media
✓Modified tinsdale
✓Cystine tellurite media
✓Pai’s coagulated egg media
232
OTHER MEDIA for Listeria monocytogenes
✓ McBride agar
✓ PEA
✓ Cold enrichment technique
233
OTHER MEDIA for Erysipelothrix rhusiopathiae
✓ BAP, tellurite, gelatin (testtube brush growth)
✓ BHIA w/ 1% glucose, 5% CO2
at 35-37C
234
OTHER MEDIA for Nocardia
✓ LJ, BHIA, SDA
235
OTHER MEDIA for C. perfringens
✓BAP = double zone hemolysis
✓Chopped meat glucose media = +gas
✓Milk media = stormy ferm.
236
OTHER MEDIA for C. tetani
BAP = swarming, faint beta hemolysis
237
OTHER MEDIA for C. botulinum
BAP = beta hemolysis
238
OTHER MEDIA for C. difficile
BAP = yellow green w/ horse stable odor
CCFA = yellow ground glass colonies
239
OTHER MEDIA for Actinomyces israelii
BHIA = molar tooth colony/ breadcrumb-like, raspberry or smooth colony
240
OTHER MEDIA for ENTEROBACTERIACEAE
✓ MAC
✓ EMB
✓ Desoxycholate agars
✓ HEA
✓ XLD
✓ SSA
✓ Desoxycholate citrate agars
***Enrichment:
Selenite F
GN broth
Tetrathionate broth
241
OTHER MEDIA for E. coli
✓MAC, EMB, XLD
✓SMAC
242
OTHER MEDIA for Edwardsiella
MAC = colorless
243
OTHER MEDIA for Shigella
✓ EMB, MAC, SSA = colorless
✓ XLD = red
✓ HEA = green to blue green
244
OTHER MEDIA for Salmonella
✓ EMB, MAC, SSA = colorless
✓ XLD, BSA = black colonies w/ metallic
silver sheen
✓ Brilliant Green agar (BGA)
✓ HEA
Enrichment: Selenite, GNB
245
OTHER MEDIA for Citrobacter
KCN medium
246
OTHER MEDIA for K. pnemoniae
EMB, MAC, XLD = +string’s test
247
OTHER MEDIA for Proteus
BAP = burnt gun odor , swarming
248
OTHER MEDIA for Y. enterocolitica
CIN
249
OTHER MEDIA for Pseudomonas aeruginosa
✓MHA
✓Pseudomonas P agar
✓Tech agar
250
OTHER MEDIA for Eikenella
CAP = corrodes, pearly sheen (mercury droplet), bleach-like odor
251
OTHER MEDIA for Flavobacterium
BAP = yellow
252
OTHER MEDIA for Haemophilus
✓ Satellite phenomenon,
dewdrop like, bleachlike
✓ CAP
✓ Levinthal and Fildes
enriched media
✓ Horseblood bacitracin by
Klein and Blazevic
253
OTHER MEDIA for Actinobacillus
actinomycetemcomitans
CAP = dots and dashes of Morse code
254
OTHER MEDIA for Pasteurella
musty or mushroom-like odor (BAP)
255
OTHER MEDIA for Francisella tularensis
✓Glucose cysteine blood agar
✓Peptone cysteine agar
✓Cystine heart agar
✓Chocolate agar
✓Rarely on Thayer Martin
256
OTHER MEDIA for Brucella
✓ Castaneda biphasic medium
✓ W or Winsconsin medium
✓ Trypticase soy agar
257
OTHER MEDIA for Bordetella
✓ Bordet-Gengou agar
✓ Jones-Kendrick charcoal agar
✓ Regan-Lowe
✓ BCYE
✓ cold casein hydrolysate
✓ Casamino acid broth
✓ Modified Stainer-Scholte agar with cyclodextrin and
cephalexin
258
OTHER MEDIA for Kingella
BAP/CAP = “fried-egg” pitting appearance
259
OTHER MEDIA for Vibrio cholerae
✓APW, TCBS,
✓Gohar, Dieudonne’s, Monsur and Aronson media
260
OTHER MEDIA for Campylobacter jejuni
✓ Butzler’s medium
✓ Skirrow’s medium (also Helicobacter)
✓ Campy Thio medium
✓ Campy-BAP medium
261
OTHER MEDIA for Leptospirae
✓Fletcher’s medium
✓Noguchi’s
✓Stewart’s
✓Ellinghausen, McCullough, Johnson and Harris (EMJH) medium
262
OTHER MEDIA for Chlamydia trachomatis
✓McCoy cells
✓C. pneumoniae
✓HeLa 229
263
OTHER MEDIA for Mycoplasma
✓SP-4 Mycoplasma medium
✓Edward-Hayflick agar
✓Shepard’s A-7B agar
264
OTHER MEDIA for Rickettsia
✓Chick embryo
265
OTHER MEDIA for Bartonella
✓ BHIA
266
OTHER MEDIA for Legionella
✓ BCYE
✓ Feeley-Gorman medium
✓ MHA w/ hemin/isovitalex
267
OTHER MEDIA for Chromobacterium
✓ smell of ammonium cyanide, violet in BAP
268
OTHER MEDIA for Gardnerella vaginalis
✓ HBBT
269
Ways to facilitate Anaerobic Cultivation
a. Special culture medium incorporated with Thioglycollate and Cystein (reducing agents)
b. Boiling of culture medium
c. Anaerobic chamber system with a vacuum pump and nitrogen gas to remove residual oxygen
d. Gas-pak jar conataining a palladium catalyst
e. Small volumes: plastic bags, pouches containing calcium carbonate and catalyst
270
Specimen for Anaerobic Cultivations is aspirated with
sterile needle and syringe
271
Anaerobic Transport Devices
➢Anaerobic Culturette
➢Bio-Bag
➢Anaerobic Pouch
➢GasPak Pouch
272
A. Anaerobic Jars
1. Brewer Jar
2. Torbal Jar
3. GasPak Jar
273
Components of Anarobic Jars
➢Hydrogen and CO2 generator envelop→ activated with water
➢Palladium-coated alumina pellets→ catalyst
➢Methylene Blue or Resazurin Indicator Strip → upon exposure to atmospheric oxygen turns blue or pink
274
→Oxygen is removed by means of electrically heated platinized catalyst with the electrical connection outside the jar
Brewer Jar
275
→uses a rubber O ring rather than Plasticine and a catalyst active at room temperature thus requiring no electrical heating
Torbal Jar
276
→most convenient and widely used anaerobic jar
→takes 30 to 45 minutes to obtain anaerobic
environment
GasPak Jar
277
Principle of Anaerobic Jars
➢With water added to the CO2 and H2 generator envelop and oxygen catalysed with H2 to water via the pellets, anaerobiosis is achieve
278
Indication of Anaerobiosis
✓Production of heat
✓Moisture inside jar
✓Decolorization of indicator strip: WHITE or COLORLESS
279
(Anaerobic Jars)
Failure to achieve anaerobic condition will result to
“poisoned” catalyst or a crack in the O ring, jar or lid
280
Jars utilizing the “Evacuation-Replacement” System
✓Air is removed by drawing a vacuum of 25 inches of mercury
✓Jar is filled with oxygen-free gas such as nitrogen between evacuations of air
✓Anaerobiosis is achieved more quickly with this method but less convenient for routine use
281
→Enclosed sytem consist of
large clear plastic, airtight
bag or chamber filled with
oxygen-free gas mixture of
nitrogen, hydrogen and CO2
→Allow materials to enter
through an air lock
Glove Box or Anaerobic Chamber
282
Anaerobiosis in Glove Box is maintained by
Palladium catalyst and Hydrogen gas
283
Components of Glove Box
a. Nitrogen Gas
→ filler for the remaining percentage of the
anaerobic straucture
b. Palladium Pellets
→ remove residual oxygen by combining with
H2 to form water
c. Silica Gel (Dessicant)
→ absorbed water
d. Methylene Blue or Resazurin
→ oxygen reduction indicator
284
Operator uses gloves or
sleeves that form airtight
seals around arms to
manipulate items
Glove Box
285
→Pre-Reduced Anaerobically Sterilized (PRAS) agar is distributed under anerobic conditions as thin layer around the inner wall of test tubes
→tubes are rolled and cooled until the melted agar forms the thin layer
Role Tube Technique
286
(Role Tube Technique)
→Syringe and needle are used through the rubber seal
Closed Method by Hungate
287
(Role Tube Technique)
→ remove the rubber stopper and insert a cannula that has oxygen-free gas flowing from the tip
Open Method
288
CULTURE MEDIA
1. Freshly made BAP
2. Enrichment media (supplemented BHIA, special Brucella Blood Agar, Laked Blood Agar)
3. Selective Media
4. Pre-Reduced Anaerobically Sterilized (PRAS)
media
5. Liquid Media
289
Selective Media for anaerobic gram (-) rods
Kanamycin-Vancomycin Blood Agar
290
Selective Media for Bacteroides melaninogenicus
Kanamycin-Vancomycin-Laked Blood Agar
291
Selective Media for Fusobacterium
and Veilonella
Neomycin-Vancomycin Blood Agar
292
Selective Media for Clostridia and anaerobic
gram(+) cocci
Neomycin Blood Agar
293
Selective Media for Clostridia
Nagler Agar (with egg yolk, Neomycin)
294
→ only medium capable of supporting
growth of 3 groups of microorganisms:
Aerobes, Microaerophiles, Anaerobes
Thioglycolate Medium
295
→reducing substance, provides low redox potential
Na Thioglycollate
296
→narrow pink layer means that the medium is reduced and can be used for anaerobic culture
→Pink layer extends to 1/3 of the medium: oxidized---boiled or autoclave to restore the narrow layer
Resazurin Indicator
297
→stored at room temperature in the dark because at refrigerator temperature it absorbs more oxygen
Thioglycollate Medium
298
Liquid Media that prevents entry of O2
Petrolatum
299
Role Tube Technique is inoculated in what 2 ways
Close Method by Hungate
Open Method
