Chemical Methods for determination of microorganism in food

Anil ShresthaMsc medical microbiology

Limulus Lysate for Endotoxins• Gram-negative bacteria are characterized by their production of

endotoxins, which consist of a lipopolysaccharide (LPS) layer (outer membrane) of the cell envelope. The LPS is pyrogenic and responsible for some of the symptoms that accompany infections caused by gram-negative bacteria.

• The Limulus amoebocyte lysate (LAL) test employs a lysate protein obtained from the blood cells (amoebocytes) of the horseshoe crab (Limulus polyphemus). The lysate protein is the most sensitive substance known for endotoxins.

• The LAL test is performed by adding aliquots of food suspensions or other test materials to small quantities of lysate preparation, followed by incubation at 37°C for 1 hr. The presence of endotoxins causes gel formation of the lysate material.

Adenosine-Triphosphate Measurement

• Adenosine-triphosphate (ATP) is the primary source of energy in all living organism. It disappears within 2 h after cell death, and the amount per cell is generally constant.

• The simplest ways to measure ATP is by use of the firefly luciferin-luciferase system. In the presence of ATP, luciferase emits light, which is measured with a liquid scintillation spectrometer or a luminometer. The amount of light produced by firefly luciferase is directly proportional to the amount of ATP added.

• The application of ATP measurement as a rapid method for estimating microbial numbers has been used in clinical microbiology.

Radiometry

• The radiometric detection of microorganism is based upon the incorporation of a ¹⁴C-labelled metabolite in a growth medium so that when the organisms utilize this metabolite, ¹⁴CO₂ is released and measured by use of a radiometry counter. For organisms that utilize glucose, ¹⁴C-glucose is usually employed. For those that cannot utilize this compound, others such as ¹⁴C-formate or ¹⁴C-glutamate are used.

Fluoro- and Chromogenic Substrates

• Some of the fluorogenic and chromogenic substrate employed in culture media iin food microbiology are

• 4-methylumbelliferyl-β-D-glucuronide (MUG)• 4-methylumbelliferyl-β-D-galactoside (MUGal)• O-nitrophenyl-β-D-galactopyranoside (ONPG)• Fluorogenic substrates are produced from the first three and

chromogenic substrates from the others. These substrates are employed in various ways in plating media, MPN broth, and for membrane filtration methods.

• MUG is the most widely used of the fluorogenic substrates, and it is hydrolysed by β-D-glucuronidase (GUD) to release the flurescent which is detectedwith long-wave ultraviolet light.