BACTERIOLOGY MTAP (PART 1)

BASIC FLOW 2 groups of clinically significant/Important

1. Gross Examination bacteria
 Observe reasons why the
1. Grown on Artificial Media
specimen must be processed
2. Not Grown on Artificial Media
 Considered as: OBLIGATE
2. Direct Microscopic Examination
INTRACELLULAR BACTERIA
 Early indicator of diagnosis
PROCESSING OF CULTURE
3. Growth and Cultivation
 Isolate and analyze 1. Select proper culture media
2. Identify temperature and atmosphere
4. Analysis of the Cultivated Organisms of incubation
 Most common: Conventional 3. Isolate for further characterization
Biochemical Testing 4. Identification of the organism

5. Identification
6. Antimicrobial Susceptibility Testing
 BACTERIOLOGY MTAP (PART 1)
TYPES OF MEDIA
TYPE EXPLANATION EXAMPLES
Supportive Supports the growth of most NONFASTIDIOUS 1. Nutrient agar
bacteria 2. Trypticase soy broth

Enrichment Contains added growth factors such as: 1. Sheep blood agar
 Blood 2. Chocolate agar
 Vitamins 3. Brain-heart infusion
 Yeast extract 4. Buffer charcoal-yeast
extract

Selective Contains additives such as: 1. Columbia colistin-

 Dyes nalidixic acid agar
 Bile salts 2. Eosin Methylene blue
 Alcohols 3. MacConkey
 Acids 4. Hektoen enteric
 Antibiotics 5. Xylose Lysine
Deoxycholate
6. Thayer Martin

Differential Formulated to provide distinct colonial 1. Eosin methylene blue

appearances based on certain biochemical 2. MacConkey
reactions 3. Hektoen enteric
 Lactose fermentation 4. Xylose Lysine
 Hydrogen sulfide production Deoxycholate

ROUTINE PRIMARY MEDIA FOR AEROBES AND FACULTATIVE ANAEROBES

MEDIUM USE COMMENTS
Bood agar, sheep Enriched medium that will  Tryptic soy broth with 5% sheep
grow most non-fastidious blood agar
bacteria  Allows differentiation of hemolysis
 All can be inoculated here

Chocolate Agar Enriched medium for  Supplies X and V factors

Hemophilus and Neisseria  Incubate in increased CO2

Columbia Colistin Selective medium for gram  Phenylethyl alcohol inhibits enteric
Nalidixic Acid agar positive cocci and anaerobic gram negative bacilli
(CNA) gram negative bacilli  Contains 5% sheep blood

Phenylethyl alcohol Selective medium for gram  Phenylethyl alcohol inhibits enteric
agar (PEA) positive cocci and anaerobic gram negative bacilli
gram negative bacilli  Contains 5% sheep blood
 BACTERIOLOGY MTAP (PART 1)
Streptococcal Selective medium for S.  Contains trimethoprim-
selective agar (SSA) agalactiae and S. pyogenes sulfamethoxazole in 5% sheep blood

Eosine Methylene Selective differential  Eosin and methylene blue inhibit

Blue (EMB) medium for isolation of gram positives
enteric gram negative bacilli  Lactose fermenters are green-black
or purple
 Non lactose fermenter = LIGHT PINK
 E. coli produces green metallic sheen
= the organism is highly lactose
fermenter
 Non lactose-fermenters are colorless

MacConkey agar Selective, differential  Bile salts and crystal violet inhibit
(MAC) medium for the isolation of most gram positives
enteric gram negative bacilli  Lactose fermenters are pink
 Non-lactose fermenters are colorless

MacConkey Sorbitol Selective medium for E. coli  E. coli O157:H7 does not ferment
agar (SMAC) O157:H7 sorbitol
 Colonies are colorless

Hektoen enteric agar Selective, differential  Bile salts, bromthymol blue, and acid
medium for the isolation of fuschin inhibits normal G1 flora
enteric pathogens from stool  Nonpathogens are orange to salmon
pink
 Nonlactose fermenters are green to
blue green
 H2S positive colonies have black
resipitate

Xylose Lysine Selective, differential  Deoxycholate inhibites many gram

deoxycholate (XLD) medium for isolation of negative rods and gram positives.
Salmonella and Shigella  4 types ofcolonies:
1. Yellow (E. coli)
2. Yellow with black centers
(some Proteus)
3. Colorless or ed colnies
(Shigella)
4. Red with black centers
(Salmonella)

Salmonella-Shigella Selective medium for  Brilliant greenand bile salts inhibit

AGAR (SS) Salmonella and Shigella coliforms
 Salmonella and shigella do not
ferment lactose – colonies are
colorless
 BACTERIOLOGY MTAP (PART 1)
 Salmonella produces H2S – black
center

BROTHS
Gram negative broth Selective enrichment  Deoxycolate and citrate salts retard
(GN) medium for isolation of growth of gram-positives
salmonella and shigella from  Subculture onto selective differential
stools and rectal swabs. agar after 6-8 hours of incubation

Selenite broth Enrichment broth used for  Subculture to enteric media after 8-
recovery of Salmonella from 12 hours of incubation
stool.
Tetrathionate Broth Enrichment broth for  Bile salts and sodium thiosulfate
recovery of Salmonella from inhibit gram positives and
stool. Enterobacteriaceae
 Inhibits most Shigella
 Should not be used for recovery of S.
typhi
 Subculture to enteric media after 12-
24 hours of incubation.

Campylobacter blood Selective enrichment  Incubate plates in increased CO2 at

agar (Campy BA) medium for isolation of 42 degrees Celsius.
Campylobacter from stool

NEISSERIA COMMON CULTURE MEDUM

Modified Thayer Selective enrichment  Vancomysin, Colistin, Nystatin, and
Martin (TM) medium for recovery of Trimethoprim inhibit growth of
NEISSERIA GONORRHOEAE other bacteria and fungi
and NEISSERIA  Incubate in increased CO2
MENINGITIDIS from  Some N. GONORRHOEAE may be
specimens with normal flora inhibited

Martin Lewis Selective enrichment  Similar to Thayer Martin but

medium for recovery of different antibiotics
NEISSERIA GONORRHOEAE  Inhibits yeast better
and NEISSERIA  Incubate in increased CO2
MENINGITIDIS from
specimens with normal flora

New York City Selective enrichment  Incubate in increased Co2

Medium medium for isolation of  Some N. GONORRHOEAE re
NEISSERIA GONORRHOEAE inhibited by antibiotics
and NEISSERIA  Genital mycoplasms will grow also.
MENINGITIDIS from
specimens with normal flora
 BACTERIOLOGY MTAP (PART 1)
MEDIA CULTURE FOR ANAEROBES
MEDIUM USE
Bacteriodes bile-esculin agar (BBE)  Selective differential medium for
isolation and identification of
BACTEROIDES FRAGILIS
 Bile salts and gentamycin act as inhibitors
 Hydrolysis of esculin indicated by
blackening of agar

Blood agar, anaerobic, CDC  Enrichment medium for isolation of

fastidious anaerobes
 Contains yeast extract: L- Cysteine,
Hemin, Vitamin K.
 Nonselective

Cycloserine cefoxitin fructose egg yolk agar  Selective medium for CLOSTRIDIUM
(CCFA) DEFFICILE
Cooked meat medium  For isolation of anaerobes, especially
pathogenic CLOSTRIDIUM
Egg-yolk agar (EYA)  For determinantion of Lecithinase and
Lipase production by clostridia and
fusobacterial
Laked kanamycin-vancomycin blood agar (LKV)  Selective medium for BACTEROIDES and
PREVOTELLA
Pheylethyl alcohol agar (PEA)  Inhibits enteric gram negative bacilli and
swarming by some clostridia
Thioglycollate broth (THIO)  All purpose medium that supports the
growth of most aerobes and anaerobes.
 Can be used as a backup broth to detect
microorganisms present in small
numbers or anaerobes,
 Thioglycolate acts as a reducing agent
 Aerobes grow at top, strict anaerobes at
bottom, facultative anaerobes
throughout.
 Store at room temperature and boil and
cool prior to use.

SPECIAL BACTERIOLOGIC MEDIA

MEDIUM USE COMMENTS
Cystine-tellurite blood agar Differential medium for  C. DIPTHERIAE produces
isolation of CORYNEBACTERIUM black colonies
DIPTHERIA
Loeffler medium Enrichment medium for  Promotes development
 BACTERIOLOGY MTAP (PART 1)
recovery and identification of of characteristic
CORYNEBACTERIUM DIPTHERIA granules.
Bismuth sulfide agar Selective medium isolation of  Bismuth sulfide and
SALMONELLA brilliant green inhibit
most others
 S. TYPHI colonies are
black surrounded by
metallic sheen
 Others are light green
 Some salmonellae may
be inhibited
Cefsulodin-irgasonovo-biocin Selective medium for YERSINIA  Crystal violet inhibits
agar (CIN) ENTEROCOLITICA, most gram negatives
AEROMONAS, and  Novobiocin inhibits
PLESIOMONAS SHIGELLOIDES gram positive cocci
 Cefsulodin inhibits most
gram positives and
gram negatives
 Y. ENTEROCOLLITICA
ferments mannitol and
appears as RED “BULL’S-
EYE” colonies
surrounded by colorless
halo.
Alkaline peptone water Enrichment medium for  Alkaline pH suppresses
recovery of VIBRIO and commensals
AEROMONAS from stool  Subcultured to TCBS
Thiosulfate citrate bile salts Selective for VIBRIOS  High pH inhibits most
sucrose agar (TCBS) bacteria
 V. CHOLERAE ferments
sucrose and roduces
yellow colonies
 V. PARAHAEMOLYTICUS
and V. VULNIFICUS
don’t ferment sucrose
and usually produce
blue-green colonies.
Rabbit blood agar Used to speciate HAEMOPHILUS  H. HAEMOLYTICUS and
H. PARAHAEMOLYTICUS
are hemolytic
Bordet-Gengou Selective enrichment medium  Potato-glycerol based
for isolation of BORDETELLA medium enriched with
PERTUSSIS blood.
 Contaminants inhibited
by methicillin
 “Cough plate”
 BORDETELLA COLONIES
 BACTERIOLOGY MTAP (PART 1)
RESEMBLE MERCURY
DROPLETS
Regan Lowe Selective for B. PERTUSSIS  Charcoal agar
supplemented with
horse blood and
aphoterecin Beta
Buffered charcoal yeast extract Enrichment medium for  Yeast extract and L-
agar (BCYE) isolation of LEGIONELLA Cysteine enhance
growth of LEGIONELLA
 Charcoal absorbs toxic
compounds
Vaginalis agar (V Agar) Nonselective enrichment  Columbia agar with
medium for isolation of human blood
GARDNERELLA VAGINALIS  Incubate in increased
CO2 for 24-72 hours
 G. VAGINALIS produces
diffuses beta hemolysis

Human blood Tween agar Semi-selective medium for G.  Incubate in increased

VAGINALIS CO2 for 48 hours.
 Colonies show diffuse
beta hemolysis
Lowenstein-Jensen Selective enrichment medium  Contains potato flour,
used to culture MYCOBACTERIA egg, and glycerol
 Malachite green inhibits
other bacteria
 M. TUBERCULOSIS
colonies are rough and
buff
Middlebrook 7H10 and 7H11 Selective enrichment medium  Agar based isoniazid-
used to culture MYCOBACTERIA resistant strains grow
better than on egg-
based media
 Contains malachite
green to inhibit other
bacteria

Fletcher medium Enrichment medium for  Contains rabbit serum.

isolation of LEPTOSPIRA from
blood, CSF, and urine
 BACTERIOLOGY MTAP (PART 1)

SELECTION OF PRIMARY CULTURE MEDIA o Disposable inoculating loops

 Agar plates are commonly used have predetermined volume
 Broth media is limited (placed in tubes o 1:100 or 1:1000
thus differing oxygen gradients) o (colonies grown x 100 or 1000)
o Used when there’s few  SEMI-QUANTITATIVE
organisms in low concentration o Ordinary loop
o Reasonable chance of recovery o QUADRANT STREAKING
of an anaerobe
 BLOOD AGAR MEDIUM
o Used to grow any NUMBER OF COLONIES GRADING
microorganism in the specimen  4+ many, heavy growth
 MACCONKEY/EOSIN-METHYLENE BLUE o Growth is out to the FOURTH
o When we suspect gram QUADRANT
negative organisms  3+ moderate growth
 ENRICHMENT BROTH o Growth is out to the third
o Recover pathogens in a quadrant
contaminated specimen (the  2+ few or light
source of the specimen is with o Growth is in the second
normal flora) quadrant
 1+ rare
o Growth in the first quadrant
SPECIMEN PREPARATION
 HOMOGENIZATION
o Grinding of tissue
 CONCENTRATION
o When dealing with fluid
o By centrifugation or filtration
 DECONTAMINATION
o Eliminates possible normal flora
o Used in LEGIONELLAE and
MYCOBACTERIA
 INOCULATED QUANTITATIVELY:
1. Urine culture
2. Tissue from burn victims
 INOCULATED SEMI-QUANTITATIVELY
o Everything/other samples

 QUANTITATIVE
o Dilution process