media Flashcards
non selective culture media
x> Support growth of most non-fastidious organisms
Example: Tryptic Soy Agar (TSA)
selective culture media
> Support growth of one type of organism but inhibit growth of others
Example: Phenylethyl Alcohol (PEA) Agar
differential media
> Allows grouping of microbes based on demonstrated characteristics on the
media
Examples: Sheep Blood Agar, MacConkey agar
enriched media
> Contain growth factors added to nonselective media to
allow fastidious organisms to grow
Example: Chocolate Agar (CHOC)
enrichment media
> A liquid medium designed to encourage small numbers of
organisms to grow
Inoculated broth is incubated for certain time period and
then sub- cultured to isolate organism of interest
Example: Gram Negative Broth (GN Broth)
broth culture media
> Supplement to agar plates designed to detect small
numbers of aerobes, anaerobes, and microaerophiles
Example: Thioglycolate broth
routine innoculation
Routine primary plating media may include the following (Lab
dependent)
> Nonselective Agar Plate
> Enriched medium for fastidious organisms for normally sterile body
fluids or sites in which fastidious organisms are expected
> Selective and Differential medium for enteric gram-negative bacilli
> Selective medium for gram-positive organisms
> Additional selective media or enrichment broths for specific
pathogens as needed (or requested)
> Broth medium may be used as a supplement with specimens from
sterile body fluids, tissues, lesions, wounds and abscesses
blood agar ( BAP/BA)
Enriched, Non-Selective and Differential
??What makes BAP differential??- hemolysis
Nutritional base (Ex. Trypticase Soy Agar)
Enriched with blood - Most often sheep blood-5% (Rabbit,
Horse and Human blood can be used)
Cultivation of Fastidious and Non-Fastidious organisms
chocolate agar ( choc)
Enriched and Non-Selective
Nutritional base
Enriched with heated red blood cells and/or other
supplements
Cultivation of Fastidious organisms (Non-Fastidious
organisms will also grow)
macconkey agar ( MAC)
Selective and Differential
Inhibits Gram Positive while allowing Non-Fastidious Gram Negative
(Selective)
Carbohydrate – Lactose (Differential)
> Lactose Positive – Red to pink colonies surrounded by precipitated bile
> Lactose Negative – Colorless colonies
Inhibitor – Bile Salts and Crystal Violet
pH Indicator – Neutral Red (Red or Pink in Acid)
PHENYLETHYL ALCOHOL BLOOD AGAR
PEA
Selective
Isolates Gram positive organisms while inhibiting Gram
negative organisms
Contains the inhibitor – Phenylethyl Alcohol
MANNITOL SALT AGAR (MSA)
Selective and Differential
Inhibits most Gram negative and many Gram positive organisms
Carbohydrate – Mannitol
Inhibitor – 7.5% salt
pH Indicator – Phenol red
Differentiates S. aureus from other species (utilization of the
Mannitol which turns the media yellow)
MUELLER HINTON AGAR (MH)
Non-Selective
Supplements can be added (ex. Salt, sheep blood, lysed
horse blood)
Used for antimicrobial susceptibility test
COLISTIN NALIDIXIC ACID (CNA)
Selective and Differential
Blood agar with additives
Inhibitors – Colistin and Nalidixic Acid
Isolates many Gram Positive while inhibiting Gram Negative
organisms
MODIFIED THAYER MARTIN (MTM)
Selective and Enriched
Mueller Hinton agar with 5% Chocolate Sheep Blood
Antibiotic additives
Isolation of Neisseria spp. (Gonococcal Selective Agar)
Also supports the growth of some other organisms
(Brucella spp./Franciscella tularensis
Sorbitol MacConkey (SMAC)
> Selective and Differential
Isolation of E. coli O157:H7 while inhibiting Gram Positive
Organisms
Carbohydrate – Sorbitol (Differential)
Inhibitor – Bile Salts and Crystal Violet
pH Indicator – Neutral Red (Red or Pink in Acid)
Sorbitol Negative - Colorless Colonies (O157:H7)
Sorbitol Positive – Red Colonies (Other E. coli)
Salmonella-Shigella (SS)
> Moderately Selective and Differential
Inhibits Gram Positive and many Gram Negative
(Selectiveness)
**Inhibits some strains of Shigella
Carbohydrate – Lactose (Differential)
Inhibitors – Bile Salts, Citrate, Brilliant Green
pH Indicator – Neutral Red (Red to Pink in acid)
H2S System
> Lactose Positive – Red-Pink Colonies (ex. E. coli/Klebsiella)
Lactose Negative (H2S Negative) – Colorless Colonies (ex.
Shigella)
H2S Positive (Lactose Negative) – Colorless Colonies with Black
Centers (ex. Salmonella/Proteus)
Hektoen Enteric (HE)
> Moderately Selective and Differential
Inhibits Gram Positive and some Gram Negative
(Selectiveness)
Carbohydrates – Lactose, Sucrose, Salicin (Differential)
Inhibitor – Bile Salts
pH Indicator – Acid Fuchsin and Bromthymol Blue (Media
turns yellow/orange/salmon pink in Acid)
H2S System
> Lactose Positive – Yellow to Salmon Color Colonies (ex. E.
coli/Klebsiella)
Lactose Negative (H2S Negative) – Colorless (green) Colonies (ex.
Shigella)
> H2S Positive (Lactose Negative) – Colorless (blue-green) Colonies with
Black Centers (ex. Salmonella)
***Proteus – Lactose Negative but some strains metabolize sucrose
and/or salicin therefore may be Blue-Green or Salmon Color on HE
specimen prep ( types )
Most specimens arrive in the laboratory in one of three forms:
> Swab
> Tissue
> Fluid
Directly inoculate onto selected media
> Pus, Urine, Sputum, Sterile body fluids
specimen prep (concentration )
Large volumes of sterile body fluids may be concentrated
and filtered to improve yield
> Fluid greater than 1 mL
- Centrifuge 20 minutes at 3000×g
- Filter if fluid is thin enough
> Resulting sediment is used to inoculate media and to
make direct specimen smears for staining/microscopic
examination
EX. CSF
specimen prep ( swabs)
> Ideally two swabs submitted
- One for direct smear
- One for culture
Swab for culture may be placed in 0.5 to 1.0 mL of
sterile broth or saline and vortexed
Material on swab breaks loose and can then be
transferred to culture media
specimen prep ( tissues )
> Tissue samples prepared by homogenization
Grind up tissue for culture before transferring to culture
media
isolation streak
> Four quadrants
> Allows grading of relative concentration of organisms
quantitative isolation streak
> Loops with specific volumes are streaked down the center
Center spread over the area of the plate
(christmas tree)
culture workup considerations
Considerations
> What is the specimen source?
> Does this source have normal biota (flora)?
> If normal biota, what do they look like?
> What are the most likely pathogens?
> What is the colony morphology for these pathogens?
> Which media is demonstrating growth, and what is the purpose of the media?
> Does it require a full workup to genus and species?
Other Considerations
> Timely, cost-effective, available resources, assuring quality laboratory results,
optimal patient care
culture workup ( routine & non routine )
Routine Specimens
> Laboratories generally have standardized processing procedures
established for routine specimens
Non-Routine Specimens
> Laboratories may receive nonroutine specimens for which there
are no standard operating procedures in place
> For these situations, the lab must use a standardized thought
process to ensure that the specimens can be cultured in an appropriate
fashion
Non- routine specimens ( points to consider)
Non-Routine Specimens - Points to Consider
> Is the specimen likely to contain low or high numbers of
organisms?
> If low, concentration of specimen is advantageous from a
large amount of specimen
> If extremely low, how important is it to enhance them? -
Such as the presence of one organism in a specimen that
should be sterile?
- Use of broth is important when growth need
enhancement
Non-Routine Specimens - Points to Consider
> Are organisms likely to be fastidious or non-fastidious?
Important factors
Choice of medium, temperature, atmosphere and length of
incubation
> Is any normal biota associated with the specimen?
> Does the specimen contain any preservatives or growth
inhibitors that must be counteracted?
Non-Routine Specimens - Points to Consider
> What is a reasonable amount to culture?
> Are all areas of the specimen homogeneous or will the
portion chosen for culture affect the results?
> Is the objective to select a single agent from a mixed
culture?
- Use enriched or selective medium in such cases
> Is there a need to culture both external and internal
surfaces?
non routine specimen examples
Vein Grafts Implant Soak Solutions Multiple Lumen Catheters Perfusates (perfusion fluid) Water Samples Equipment
vascular catheter tip ( non routine)
> Ex. Vascular Catheter Tip
- Submitted for culture to aid in the diagnosis of catheter related
infections
- Maki roll technique used in many laboratories
- Using sterile forceps, a 5-7 cm segment of the catheter is rolled
across the surface of a BAP
incubation requirements
Most bacteria cultures grow between 35° C and 37° C.
Oxygen conditions depend on organisms.
> Aerobic - grow in ambient air
> Anaerobic - grow in the absence of air
> Capnophilic - requires increased CO2
> Microaerophilic - requires reduced O2 and increased CO2
Typical Incubation Times
> Most routine bacterial cultures held for 48 to 72 hours
> Cultures for anaerobes and broth cultures may be held 5
to 7 days
> Unusual organisms may require special medium or
conditions beyond the routine
