Why biochemical tests are important

Positive reactions turn the bacteria violet to purple immediately, or up to 30 seconds. Delayed reactions should be ignored. Microbiology Resource Center. Overview Lab Procedure Motility Test Fluid Thioglycolate Medium FTM Catalase Oxidase Identifying Unknown Bacteria Overview This lab should help give you the background information and techniques you will need to successfully perform general biochemical tests in order to help identify unknown bacterial samples.

You will perform general biochemical tests on an unknown organism. For each biochemical test you perform, make sure to record the following in your lab book: What does a positive test result look like?

What is the biochemical basis of the test? Lab Procedure We have included the basic procedure for doing each biochemical test in the table below. Table 1: Brief Description of general tests and probable results. These enzymes function inside the bacteria cells. They are mainly responsible for the synthesis of new protoplasmic substances and production of cellular energy from simple substances permeating into the cells through the cell membrane.

There are many intracellular enzymes, which help in the utilization of these simple substances. It is necessary for cellular survival and function and is the basis of cellular metabolism. As a result of these metabolic processes, metabolic products are formed and excreted by the cells into the environment. Assay of these end products aids in the identification of bacteria.

These are the enzymes secreted by the bacteria cells to the surrounding to hydrolyse high molecular weight or complex substances, such as proteins, polysaccharides and fats, which cannot permeate the bacterial cell membrane, because of their large size proteins and polysaccharides or because of their complex nature fats.

The extracellular enzymes hydrolyse them into their respective simple building blocks. Proteins are hydrolysed to amino acids, polysaccharides like starch to glucose and fats like triglycerides to glycerol and fatty acids.

The low molecular weight simple substances, so produced, can permeate through the cell membrane into the bacteria cells, where they are utilized with the help of the intracellular enzymes with the liberation of metabolic wastes.

The ability of a bacteria to produce these extracellular enzymes is species specific and therefore, is used for the identification of bacteria.

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View Metrics. Email alerts Article activity alert. Advance article alerts. New issue alert. In progress issue alert. Receive exclusive offers and updates from Oxford Academic. Notice that Shigella dysenteriae far left ferments glucose but does not produce gas.

Because the same pH indicator phenol red is also used in these fermentation tubes, the same results are considered positive e. The degree of hemolysis by these hemolysins is helpful in differentiating members of the genera Staphylococcus , Streptococcus and Enterococcus.

Often when inoculating a BAP to observe hemoloysis patterns, investigators will also stab several times through the agar using an inoculating loop. This stab allows for the detection of streptolysin O, a specific hemolysin produced by Streptococcus pyogenes. This hemolysin is inactivated by O 2 and is only seen subsurface in an anaerobic environment around the stab mark. Note the oval-shaped areas of clearing around the stab marks in the picture below; these are caused by streptolysin O.

This is a medium that is both selective and differential. It tests the ability of organisms to hydrolyze esculin in the presence of bile. It is commonly used to identify members of the genus Enterococcus E faecalis and E.

The first selective ingredient in this agar is bile, which inhibits the growth of Gram-positives other than enterococci and some streptococci species. The second selective ingredient is sodium azide. This chemical inhibits the growth of Gram-negatives. The differential ingredient is esculin. If an organism can hydrolyze esculin in the presence of bile, the product esculetin is formed. Esculetin reacts with ferric citrate in the medium , forming a phenolic iron complex which turns the entire slant dark brown to black.

The tube on the far right was inoculated with E. The tube in the center was inoculated with a bilie esculin negative organism and the tube on the left was uninoculated. It tests the ability of an organism to do several things: reduce sulfur, produce indole and swim through the agar be motile.

SIM is commonly used to differentiate members of Enterobacteriaceae. Sulfur can be reduced to H 2 S hydrogen sulfide either by catabolism of the amino acid cysteine by the enzyme cysteine desulfurase or by reduction of thiosulfate in anaerobic respiration. If hydrogen sulfide is produced, a black color forms in the medium. Proteus mirabilis is positive for H 2 S production. The organism pictured on the far left is positive for hydrogen sulfide production.

Bacteria that have the enzyme tryptophanase, can convert the amino acid, tryptophane to indole. Escherichia coli is indole positive. The organism pictured second from left is E. SIM tubes are inoculated with a single stab to the bottom of the tube. If an organism is motile than the growth will radiate from the stab mark and make the entire tube appear turbid.

Pseudomonas aeruginosa and the strain of Proteus mirabilis that we work with are motile. It also allows for identification of sulfur reducers. This media is commonly used to separate lactose fermenting members of the family Enterobacteriaceae e.

Escherichia coli from members that do not ferment lactose, like Shigella dysenteriae. These lactose nonfermenting enterics generally tend to be the more serious pathogens of the the gastrointestinal tract.

The first differential ingredient, glucose, is in very short supply. Organisms capable of fermenting this sugar will use it up within the first few hours of incubation. Glucose fermentation will create acidic byproducts that will turn the phenol red indicator in the media yelllow. Thus, after the first few hours of incubation, the tube will be entirely yellow. At this point, when the glucose has been all used up, the organism must choose another food source.

If the organism can ferment lactose, this is the sugar it will choose. Lactose fermentation will continue to produce acidic byproducts and the media will remain yellow picture on the far left below. If gas is produced as a result of glucose or lactose fermentation, then fissures will appear in the agar or the agar will be lifted off the bottom of the tube.

The deamination of the amino acids creates NH 3 , a weak base, which causes the medium to become alkaline. The alkaline pH causes the phenol red indicator to begin to turn red.

Since the incubation time is short h , only the slant has a chance to turn red and not the entire tube. Thus an organism that can ferment glucose but not lactose, will produce a red slant and a yellow butt in a KIA tube second from the left below.

These organisms are the more serious pathogens of the GIT such as Shigella dysenteriae. The slant of the tube will be red and the color of the butt will remain unchanged picture on the far right below.