Diagnostic Microbiology Lab

30 May - 3 June 2011-  First week of industrial training!! My first rotation at diagnostic microbiology lab. Here the very interesting things that learned from here was how to translate the knowledge of microbiology for diagnostic purposes. In order to explain what exactly I learned from this lab, I divide to 2 part which ; The principle of Gram staining and Identification hierarchy.

Principle of Gram staining

Gram staining was introduced by Danish scientist called Hans Christian Gram (1853-1938) in 1884 to distinguish between Pneumococci and Klebsella pneumonia bacteria. Until now, no others staining can replaced the Gram staining as major tools to detection of bacteria. The purposes of gram staining is to distinguish between Gram positive and Gram negative bacteria. 

Gram staining has 4 basic steps; First the application of primary stain (crystal violet) to a heat-fixed smear of bacterial culture, followed by the addition of a mordant (iodine), after that rapid decolourisation of alcohol or acetone and lastly counterstain with safranin or basic fuchsin.

How gram staining can distinguish between gram positive and gram negative bacteria. Gram positive bacteria have a thick mesh cell wall made of peptidoglican and gram negative bacteria have thin layer of peptidoglican with additional outer membrane layer made of lipid. Crystal violet (CV) dissociate in aqueous solution to form CV+ ion and Cl- ion. These ion penetrate through the peptidoglican cell wall and cell membrane of both gram positive and gram negative bacteria. The CV+ ion interact with negatively charged components of bacterial cells and stain cells purple.

Iodine (I- or I3-) interact with CV+ and form large complex of crystal violet and iodine ( CV-I). When decolourization with alcohol  is added, it interact with the lipids of cell membrane. A gram negative bacteria will lose its outer membrane and the peptidoglican layer is left exposed. The CV-I complexes are washed from the membrane. In contrast, a Gram-positive cell becomes dehydrated from ethanol treatment. The large CV-I complexes become trapped within Gram-positive cell due to the multilayer nature its peptidoglican. After the decolourisation step, the Gram-positive bacteria remains purple and the Gram-negatives loss its purple colour. Counterstain, which is usually postively charged safranin is applied last to give decolorised Gram-negative bacteria a pink or red colour.

In clinical diagnostic purpose, it  is important to distinguish between Gram-positive and Gram negative bacteria is to screen the pathogen. Most Gram-negative bacteria are pathogenic but not in Gram-positive bacteria. Gram-positive bacteria depend on species of bacteria, type of samples, and also how they colonized  part of the body. If certain bacteria colonized to much in one area of the body, it may due to immunodeficiency that cause recurrent bacteria infection. After we identify whether it Gram-positive or Gram negative bacteria, to identify the specific species of bacteria we followed the identification hierarchy.

Bacteria Identification Hierarchy


After we stain bacteria via Gram staining and observe under the microscope, we can distinguish whether this is Gram positive bacteria or Gram negative bacteria. After that we followed the bacteria identification hierarchy to screen which specific bacteria species whether its pathogenic or not? Bacteria identification hierarchy can be divided by two which Gram positive  bacteria identification and gram negatives identification  hierarchy:

The figure above shows the identification hierarchy for gram-positive bacteria. By observing the gram-staining of bacteria culture under microscope, if the bacteria cultured stained purple, we observed its morphologwhether it shape cocci or bacilli, if cocci we can further down the hierarchy whether its Staphylococcus or Streptrococcus. So Catalase test must be done to both of them. The principle of  catalase test is all the Staphylococcus bacteria have catalase enzyme with act on hydrogen peroxide to release oxygen.  In this test, we can observed the immediate bubbles.

After we know the bacteria is Staphylococcus, we further down the hierarchy, at this point this is very important. Because we must do Coagulase test to distinguish whether the bacteria culture is the Staphylococcus aureus or not. S.aureus is well known dangerous pathogenic bacteria. Because the bacteria culture from patient sample, this is important to know whether the patient have S.aureus or not. The principle of coagulase tests is to know whether the bacteria have coagulase enzyme or not? S.aureus contain coagulase enzyme which catalyze the formation of fibrin clot in plasma. In this test we can observed the clotting/ clumping formation

The figure below is the gram negative bacteria identification hierarchy. As mentioned above, most of gram negatives bacteria are pathogenic. To identify the specific gram neagtive bacteria, just follow the identification hierarchy.

Both Gram-staining and Identification Hierarchy is the basic knowledge of diagnostic microbiology. Actually that has more diagnosis. For example, biochemical test and also sensitivity test. Right now, the more fast and accurate in bacteria identification that available in kit such as API test kit. As conclusion, what I learned from the industrial training in diagnostic microbiology laboratory is how I see the very simple Gram staining principle  is very useful in diagnosis of diseases. Furthermore, in analysis what exactly bacteria infect the patient, besides we analyse what the type of bacteria  in patient's samples, we must know the medical history of patient and also we must observed whether the bacteria is colonized enough in patient body to be considered its pathogenic.