APT Agar and Broth

Intended Use

APT Agar is used for cultivating heterofermentative lactobacilli and other organisms requiring high thiamine content. It is also used for maintaining stock cultures of Weissella (Lactobacillus) viridescens ATCC™ 12706 used in the assay of thiamine. APT Broth is used for culturing Weissella viridescens ATCC 12706 used in the assay of thiamine. It is also used for cultivating heterofermentative lactobacilli and other organisms requiring high thiamine content.

Summary and Explanation

Evans and Niven1 investigated cultivating the heterofermentative lactobacilli that cause the faded or greenish discoloration of cured meat products, while Deibel, Evans and Niven2 investigated thiamine requiring bacteria, specifically Lactobacillus viridescens. Their formulations led to the development of APT Agar and APT Broth.

Historically, the lactic acid bacteria, a group of acid-producing bacteria, included the genera Streptococcus, Leuconostoc, Pediococcus and Lactobacillus; currently, taxonomists include
a number of additional genera (e.g., Weissella).3 These organisms are widespread in nature and are associated with bacterial spoilage of foods such as dairy, meat and vegetable products.3 One use of APT Agar and APT Broth is for cultivating these heterofermentative lactic acid bacteria from food products.3

APT Agar and APT Broth are also used in the microbiological assay of thiamine. In the assay, APT Agar is the maintenance medium that preserves the viability and sensitivity of Weissella
viridescens ATCC 12706. APT Broth is used for growing Weissella viridescens ATCC 12706 and preparing the inoculum.

Principles of the Procedure

APT Agar and APT Broth contain peptone as a source of carbon, nitrogen, vitamins and minerals. Yeast extract supplies B-complex vitamins which stimulate bacterial growth. Dextrose
is the carbohydrate. The manganese chloride, magnesium sulfate and ferrous sulfate provide ions used in replication by lactobacilli. Polysorbate 80 is a source of fatty acids required
by lactobacilli. Agar is the solidifying agent in APT Agar.

User Quality Control

Identity Specifications
APT Agar
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution:                       6.12%, soluble in purified water upon boiling.
                                    Solution is medium amber, clear to slightly
                                    opalescent, may have a slight precipitate.
Prepared Appearance:    Medium amber, clear to slightly opalescent, may
                                   have a slight precipitate.
Reaction of 6.12%
Solution at 25°C:           pH 6.7 ± 0.2
APT Broth
Dehydrated Appearance: Light tan, free-flowing, homogeneous.
Solution:                       4.62%, soluble in purified water with slight
                                   heating. Solution is opalescent when hot. After
                                   cooling, is light to medium amber, clear to
                                   very slightly opalescent, may have a slight
                                   precipitate.
Prepared Appearance:   Light to medium amber, clear to very slightly
                                  opalescent without significant precipitate.
Reaction of 4.62%
Solution at 25°C:          pH 6.7 ± 0.2
Cultural Response
APT Agar or APT Broth
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 24-48 hours.

Formula

APT Agar
Approximate Formula* Per Liter
Yeast Extract................................................................ 7.5 g
Pancreatic Digest of Casein.......................................... 12.5 g
Dextrose.................................................................... 10.0 g
Sodium Citrate............................................................. 5.0 g
Thiamine Hydrochloride............................................... 1.0 mg
Sodium Chloride.......................................................... 5.0 g
Dipotassium Phosphate................................................ 5.0 g
Manganese Chloride................................................... 0.14 g
Magnesium Sulfate...................................................... 0.8 g
Ferrous Sulfate........................................................... 0.04 g
Polysorbate 80............................................................. 0.2 g
Agar.......................................................................... 15.0 g
APT Broth
Consists of the same ingredients without the agar.
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

APT Agar
1. Suspend 61.2 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.
3. Autoclave at 121°C for 15 minutes. Avoid overheating.
4. Test samples of the finished product for performance using stable, typical control cultures.
APT Broth
1. Suspend 46.2 g of the powder in 1 L of purified water. Mix thoroughly.
2. Warm slightly to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes. Avoid overheating.
4. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

For maintaining stock cultures of Weissella viridescens ATCC 12706 prepare a stab inoculation. Prepare stock cultures in triplicate at monthly intervals. One of the transfers is saved for the preparation of stock cultures. The others are used to prepare inoculum in APT Broth for assay as needed. Following incubation at 35-37°C for 24-48 hours, store stock cultures at 2-8°C.

Expected Results

Refer to appropriate references and procedures for results.

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