Lactose Lecithin Agar

Intended Use:

Recommended for isolation and differentiation of histotoxic Clostridia from clinical specimens.

Composition**

Final pH (at 25°C): 6.8±0.2

**Formula adjusted, standardized to suit performance parameters

# Equivalent to Pancreatic digest of heart muscles

Directions

Suspend 58.48 grams in 1000 ml purified/distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Mix well and pour into sterile Petri plates.

Principle And Interpretation

Clostridium species are widely distributed in nature and are also associated with humans, either as non-pathogens at a variety of anatomic locations or at infected sites. Diseases caused by members of the genus Clostridium generally fall into one of the three categories: a) non-invasive disease in which toxin(s) is responsible for all the symptoms. b) invasive (histotoxic) disease in which a progressive infections process and tissue destruction occur and c) purulent disease in which a closed-space mixed infection involving multiple organisms is present (1).

Histotoxic clostridia can be isolated on egg yolk containing medium, as demonstrated by McClung and Toabe (2). This medium was further supplemented with additional milk and lactose to differentiate clostridia on the basis of lecithinase production, casein hydrolysis and lactose fermentation (3). Selectivity was obtained by the incorporation of neomycin sulphate. Subsequently, eggs were replaced by purified lecithin, to obtain an egg-free medium (4). This egg-free medium was further modified with reduced concentration of neomycin and additional sodium azide, which enhanced the selective properties of the medium (5). This refined medium was designated as Lactose Lecithin Agar, which is used for isolation and differentiation of histotoxic clostridia from clinical specimens.

Tryptone, Peptone and HM hydrolysate provide carbonaceous and nitrogenous compounds essential for the growth of bacteria. Lactose is the fermentable carbohydrate with bromocresol purple being the pH indicator. L-cysteine helps to create anaerobic conditions. Yeast extracts supplies vitamin B-complex nutrients. Corn starch neutralizes toxic fatty acids if any, present in the medium. Neomycin and sodium azide inhibit accompanying gram-negative and gram-positive organisms.

Type of specimen

Clinical- stool, abscess

Specimen Collection and Handling

Before inoculation, prepared media plates should be pre-reduced by placing under anaerobic conditions for 18-24 hours. Specimens should be inoculated on these pre-reduced plates. A non-selective media should be inoculated simultaneously (1,6). An opalescent zone surrounding the colonies indicates lecithinase production. Yellow colour around colonies indicates lactose fermentation. After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

In Vitro diagnostic use only. For professional use only. Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

1. Ensure that the clinical samples are properly transported under anaerobic conditions.
2. Proper anaerobic conditions must be maintained for optimal recovery of organisms

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance: Cream to yellow homogeneous free flowing powder

Gelling: Firm, comparable with 1.5% Agar gel

Colour and Clarity of prepared medium: Light purple coloured slightly opalescent gel forms in Petri plates

Reaction: Reaction of 5.85% w/v aqueous solution at 25°C. pH: 6.8±0.2

pH: 6.60-7.00

Cultural Response: Cultural characteristics observed under anaerobic condition, after an incubation at 35-37°C for 48 hours.

Key:

(#)- Formerly known as Clostridium sordellii

Storage and Shelf Life

Store dehydrated and the prepared medium at 2-8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (7,8).

Reference

1. Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Eds.), 2003, Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology, Washington, D.C.
2. McClung L. S. and Toabe R., 1947, J. Bacteriol., 53:139.
3. Willis A. T. and Hobbs G., 1959, J. Pathol. Bacteriol., 77:511.
4. Willis A. T., 1960, J. Pathol. Bacteriol., 80:379.
5. Ellner P. D. and O. Donnell D., 1971, Am. J. Clin. Pathol., 56:197.
6. Willis A. T. and Hobbs G., 1959, J. Pathol. Bacteriol., 77:511.
7. Isenberg (Ed.), 1992, Clinical Microbiology Procedures Handbook, American Society for Microbiology, Washington, D.C.
8. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.