Download Tinsdale Agar Base Tinsdale Enrichment Desiccated

Section III
T
Thiotone E Peptone, cont.
User Quality Control
Identity Specifications
BBL™ Thiotone™ E Peptone
Dehydrated Appearance:
Tan, fine, homogeneous, free of extraneous material.
Solution:
2.0% solution, soluble in purified water. Solution is clear to moderately hazy.
Reaction of 2.0%
Solution at 25°C:
pH 6.5-7.5
Cultural Response
Biochemical Reactions
BBL™ Thiotone™ E Peptone
Prepare a sterile solution of Thiotone E Peptone as directed below. Adjust final pH to 7.2-7.4. Inoculate and incubate at 35 ± 2°C for 18-48 hours.
TEST
TEST SOLUTION
ORGANISM
ATCC™
INOCULUM CFU
RESULT
Fermentable Carbohydrates
Indole Production
Acetylmethylcarbinol
Production
Hydrogen Sulfide Production
2%
0.1%
0.1% with
0.5% dextrose
1%
Escherichia coli
Escherichia coli
Enterobacter
aerogenes
Citrobacter freundii
29552
29552
13048
~107
0.1 mL, undiluted
0.1 mL, undiluted
Positive
Positive
Positive
8454
0.1 mL, undiluted
Positive
Growth Response
BBL™ Thiotone™ E Peptone
Prepare a sterile solution of peptone agar without (plain) and with 5% sheep blood (SB) using 10 g of Thiotone E Peptone, 2.5 g of sodium chloride and
6.5 g of agar in 500 mL of purified water. Adjust final pH to 7.2-7.4. Inoculate and incubate plates at 35 ± 2°C for 2-3 days (incubate streptococci with
3-5% CO2).
ORGANISM
ATCC™
INOCULUM CFU
RECOVERY PLAIN
RECOVERY WITH SB
HEMOLYSIS
Enterococcus faecalis
Streptococcus pneumoniae
Streptococcus pyogenes
29212
6305
49117
103-104
103-104
104-105
Good
N/A
Good
N/A
Good
Good
–
Alpha
Beta
Directions for Preparation from
Dehydrated Product
Refer to the final concentration of Thiotone E Peptone in the
formula of the medium being prepared. Add product as
required.
Procedure
See appropriate references for specific procedures using
Thiotone E Peptone.
Expected Results
References
1. Tortora. 1984. Appl. Environ. Microbiol. 47:1172.
2. Clesceri, Greenberg and Eaton (ed.). 1998. Standard methods for the examination of water and
wastewater, 20th ed. American Public Health Association, Washington, DC.
3. Kwinn. 2001. Bug Journal, Biology Department, Massachusetts Institute of Technology. 4:193.
4. Horowitz (ed.). 2000. Official methods of analysis of AOAC International, 17th ed. AOAC
International, Gaithersburg, Md.
5. U.S. Food and Drug Administration. 1995. Bacteriological analytical manual, 8th ed. AOAC
International, Gaithersburg, Md.
Availability
BBL™ Thiotone™ E Peptone
AOAC BAM SMWW
Cat. No.
212302
Dehydrated – 500 g
Refer to appropriate references and procedures for results.
Tinsdale Agar Base
Tinsdale Enrichment Desiccated
Intended Use
Tinsdale Agar Base is used with Tinsdale Enrichment Desiccated in isolating and differentiating Corynebacterium
diphtheriae.
Summary and Explanation
Tinsdale Agar Base, supplemented with Tinsdale Enrichment,
is employed in the cultural diagnosis of diphtheria. Diphtheria, an acute infectious disease primarily of the upper respira562
tory tract but occasionally of the skin,1 is caused by toxigenic
strains of Corynebacterium diphtheriae. The three biotypes
are mitis, intermedius and gravis.1 The signs and symptoms of
the disease are a pharyngeal membrane, sore throat, malaise,
headache and nausea.2 Death can result from respiratory
obstruction by the membrane or myocarditis caused by the toxin.2
Tinsdale3 developed a serum-cystine-thiosulfate-tellurite agar
medium for the primary isolation and differentiation of
Tinsdale Agar Base, cont.
Uninoculated
Plate
User Quality Control
Corynebacterium diphtheriae
ATCC™ 8028
Identity Specifications
Difco™ Tinsdale Agar Base
Dehydrated Appearance:
Light beige, free flowing, homogeneous.
Solution:
4.5% solution, soluble in purified
water upon boiling. Solution is light to
medium amber, slightly opalescent to
opalescent.
Prepared Appearance:
Light to medium amber, slightly
opalescent to opalescent.
Reaction of 4.5%
Solution at 25°C:
pH 7.4 ± 0.2
Difco™ Tinsdale Enrichment Desiccated
Desiccated Appearance:
Light to dark tan cake; variations may
occur.
Solution:
Soluble in purified water. Solution is
light to dark amber, clear to opalescent,
may have a slight precipitate.
Cultural Response
Difco™ Tinsdale Agar Base with Tinsdale Enrichment
Desiccated
Prepare the medium per label directions. Inoculate to obtain discrete
colonies and stab several times using an inoculating needle; incubate at
35 ± 2°C for 18-48 hours.
ORGANISM
Corynebacterium diphtheriae
biotype gravis
Corynebacterium diphtheriae
biotype mitis
Klebsiella pneumoniae
Streptococcus pyogenes
ATCC™
INOCULUM CFU
RECOVERY
APPEARANCE
8028
102-103
Good
Brown with halos
8024
13883
102-103
102-103
Brown with halos
–
19615
102-103
Good
Marked to complete
inhibition
Poor to fair
C. diphtheriae. This formulation distinguished between
C. diphtheriae and diphtheroids which exhibited similar
characteristics. The differential principle is based on the
capacity of C. diphtheriae to produce a brown or black halo
around the colonies.
Billings4 simplified Tinsdale Basal Medium by using Proteose
Peptone No. 3 as a nutrient source. This modification improved
the differential qualities and recovery of C. diphtheriae. Tinsdale
Agar Base and Tinsdale Enrichment are prepared according to
the Billings4 modification. Moore and Parsons5 confirmed
the halo formation of C. diphtheriae with one exception;
C. ulcerans occasionally produced colonies similar to
C. diphtheriae and required biochemical identification.
Tinsdale Enrichment Desiccated contains bovine serum, sodium
hydroxide, L-cystine, sodium thiosulfate and potassium
tellurite in the quantity and proportion described by Billings.4
Principles of the Procedure
Peptone provides the nitrogen, vitamins, carbon and amino
acids in Tinsdale Agar Base. Sodium chloride maintains the
osmotic balance of the medium. Agar is the solidifying agent.
Tinsdale Enrichment contains bovine serum, which provides
essential growth factors. Sodium hydroxide maintains the pH.
T
Brown to black without halos
L-cystine and sodium thiosulfate are H2S indicators. Potassium
tellurite is a selective agent. The formation of black to brown
halos surrounding the colony results from the reduction of
potassium tellurite to metallic tellurite.
Stabbing the medium with an inoculating needle accentuates
darkening of the medium by C. diphtheriae.
Formulae
Difco™ Tinsdale Agar Base
Approximate Formula* Per Liter
Proteose Peptone No. 3 ........................................... 20.0
Sodium Chloride ........................................................ 5.0
Agar ......................................................................... 20.0
g
g
g
Difco™ Tinsdale Enrichment Desiccated
Contains Bovine Serum, L-Cystine, Sodium Hydroxide, Sodium
Thiosulfate and Potassium Tellurite at pH 8.0-10.0.
*Adjusted and/or supplemented as required to meet performance criteria.
Directions for Preparation from
Dehydrated Product
Difco™ Tinsdale Agar Base
1. Suspend 45 g of the powder in 1 L of purified water. Mix
thoroughly.
2. Heat with frequent agitation and boil for 1 minute to
completely dissolve the powder.
563
Section III
T
Tinsdale Agar Base, cont.
3. Dispense 100 mL amounts into flasks.
4. Autoclave at 121°C for 15 minutes.
5. Aseptically add 15 mL rehydrated Tinsdale Enrichment to
each 100 mL at 50-55°C. Mix well.
6. Test samples of the finished product for performance using
stable, typical control cultures.
Difco™ Tinsdale Enrichment Desiccated
1. Rehydrate with 15 mL sterile purified water.
2. Rotate in an end-over-end motion to dissolve completely.
Procedure
1. For a complete discussion on the collection, isolation and
identification of C. diphtheriae and other Corynebacterium
species, refer to the appropriate procedures outlined in the
references.1,2,6
2. Inoculate plates with the test organisms in a manner to
obtain discrete colonies and stab the medium several times
with an inoculating needle.
3. Definitive identification of a strain of C. diphtheriae as a
true pathogen requires demonstration of toxin production.6
Characteristic colonies of C. diphtheriae may be inoculated
directly onto KL Virulence Agar enriched with KL
Virulence Enrichment and containing Taxo™ KL Antitoxin
Strips for toxigenicity tests.
Expected Results
The appearance of brown-black colored colonies surrounded
by brown-black halos is presumptive evidence for
C. diphtheriae.1
Limitations of the Procedure
1. Tinsdale Agar is not suitable as a primary plating medium,
since it may not support the growth of some strains of
C. diphtheriae.1
2. C. ulcerans, C. pseudotuberculosis and (rarely) Staphylococcus species may produce a characteristic halo on Tinsdale
Agar.1
3. Do not read Tinsdale Agar early because several organisms
may exhibit slight browning on this medium in 18 hours.1
4. Incubation in 5-10% CO2 retards the development of
halos on Tinsdale Agar.1
5. On media containing tellurite, diphtheria bacilli are shorter
and stain more uniformly; however, granules are less readily
observed than when grown on Loeffler’s medium.7
6. Further biochemical tests may be necessary to distinguish
between C. diphtheriae and C. ulcerans due to similar
reactions on this medium.
References
1. Isenberg (ed.). 1992. Clinical microbiology procedures handbook, vol. 1. American Society for
Microbiology, Washington, D.C.
2. Funke and Bernard. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical
microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
3. Tinsdale. 1947. J. Pathol. Bacteriol. 59:461.
4. Billings. 1956. An investigation of Tinsdale Tellurite medium: its usefulness and mechanisms of
halo-formation. M.S. thesis. University of Michigan, Ann Arbor, Mich.
5. Moore and Parsons. 1958. J. Infect. Dis. 102:88.
6. Forbes, Sahm and Weissfeld. 1998. Bailey & Scott’s diagnostic microbiology, 10th ed. Mosby, Inc.,
St. Louis, Mo.
7. Bailey and Scott. 1966. Diagnostic microbiology, 2nd ed. The C. V. Mosby Company, St. Louis, Mo.
Availability
Difco™ Tinsdale Agar Base
Cat. No.
278610
Dehydrated – 500 g
Difco Tinsdale Enrichment Desiccated
™
Cat. No.
234210
Tube – 6 × 15 mL*
*Store at 2-8°C.
Bacto™ Todd Hewitt Broth • Todd Hewitt Broth
Todd Hewitt Broth with Gentamicin and Nalidixic Acid
Intended Use
Todd Hewitt Broth is a general-purpose medium, which
primarily is used for the cultivation of beta-hemolytic streptococci, especially for serological studies.
Todd Hewitt Broth with Gentamicin and Nalidixic Acid is used
for the selective enrichment of group B streptococci (Streptococcus agalactiae), especially from genital specimens.
Summary and Explanation
Todd Hewitt broth originally was developed for use in the
production of streptococcal hemolysin.1 The modification of
Updyke and Nickle2 is used for the growth of beta-hemolytic
streptococci for use in fluorescent antibody test procedures3
and for serological typing based on the production of
type-specific M protein.4
564
Since its emergence in the 1970s, neonatal group B streptococcal
disease has become the major infectious cause of illness and
death among newborns. Prior to 1994, an estimated 7,600
episodes of invasive group B streptococcal disease, primarily
sepsis and meningitis, occurred in newborns each year in the
United States, with approximately 80% of those episodes
representing early-onset disease occurring within the first week
of life.5 The disease is spread to newborns through vertical
transmission from a mother who carries group B streptococci
in her anorectum or genital tract.
The Centers for Disease Control and Prevention (CDC) has
published guidelines for screening and use of intrapartum
chemoprophylaxis for prevention of neonatal group B
streptococcal disease.6 The use of Todd Hewitt Broth with
Gentamicin and Nalidixic Acid (or Lim Broth) is recommended
to maximize the likelihood of recovering group B streptococci
upon plating on sheep blood agar.