IDENTIFICATION OF UNKNOWN BACTERIAL ORGANISMS

IDENTIFICATION OF UNKNOWN BACTERIAL ORGANISMS

INTRODUCTION

     The isolation and identification of bacteria is an essential diagnostic tool in the study of microbiology especially in the investigation of pathogenic bacteria that causes infectious diseases.

       In this chapter we shall introduce a variety of biochemical methods and tools to isolate and identify unknown bacteria based on characteristic gene sequences. The biochemical test of microorganisms relies on enzyme which is “glycoprotein”. Enzymes are important biosynthetic chemical found in all organisms. The kind of enzyme produced by an organism dictate the extent of its biosynthetic capabilities.

       An unknown organism say Q was prepared by the lab scientist for purpose of identification. The bacteria strain can be identified using the following biochemical processes.

       IMViC (Indole, Methyl Red, Voges-Proskauer, Citrate) refers to different tests. Indole test determines the ability of the organism to cleave the amino acid Tryptophan into indole, the presence of indole was detected by addition of Kovac’s reagent. Methyl Red test is an important test to differentiate between glucose fermenting Enterobactericae in which red indicates positive with pH below 4.5. Citrate test shall indicate the ability of an organism to utilize citrate by changing from green to blue. Hydrogen peroxide (H₂S) breaks catalase producing bacteria by giving off bubbles. Physiological characteristics are obtained by oxidative and fermentation test.

         Interestingly, bacteria produce gasses during fermentation process which can be used by addition of inverted tubes (Durham tubes). TSIA is differential medium that contain lactose, sucrose and small quantity of glucose. It is used to differentiate enteric organism which has the ability to reduce sulphur and ferment carbohydrate. Gas production is detected by holes in the agar while H₂S production is seen by blackening of the medium due to iron present and combining with hydrogen sulphide to form ferrous sulphide.

       Enteric Flora makes its abode within the lumen of the intestine consisting mainly of organism of the phyla bacteriodetes and firmicutes e.g Skin Flora.

      Enteric pathogens – These are disease causing organisms e.g intestinal bacteria; capable of causing disease to animals and human. E.g bacteria, viruses, etc

       Enteric disease description – These are diseases that infect an organism or body mainly as a result of taking contaminated food or water, they can enter the body through the mouth to the intestinal system.

        Purpose of identification – Organisms are identified for the purpose of proper classification.

MATERIALS/REAGENTS/MEDIA NEEDED

-Unknown slant culture (Organism Q)

-  Kovac's reagent

-  Simmons citrate agar

-  1 MR-VP broth (Glucose Phosphate)

- 1 filter paper soaked with oxidase reagent

-  1 inoculating needle

- 1 inoculating wire loop

- 1 Starch agar plate

-  Triple Sugar Iron Agar slant (TSIA)

-  1 test tube of glucose broth

-  1 test tube of lactose broth

-  1 sterile test tube

-  1 test tube rack

- 3% hydrogen peroxide

-  1 motility agar test tube

-  Bunsen burner

-  1% peptone water

-  16% Alpha-Naphthol

-   40% Potassium Hydroxide (KOH)

METHODOLOGY

·      
          OXIDASE TEST

   -     The workbench should be          disinfected.

   -      The wire loop should be sterilized.

   -    The organism (Q) should be smeared on the filter paper which should be placed on the petri dish containing oxidase reagent using a wire loop.

   - It should be incubated for 24-48 hours.

·        CATALASE TEST

-         3% Hydrogen peroxide should be dropped on the slide.

-         The wire loop should be sterilized and allowed to cool.

-         Using the wire loop, the organism (Q) should be collected and smeared on the Hydrogen peroxide placed on the slide.

·        MOTILITY TEST

-         The inoculating needle should be sterilized and used to take the organism (Q).

-         Use the needle to stab in the motility tube.

-         Inoculated for 24-48 hours.

·        STARCH HYDROLYSIS TEST

-         Sterilize the wire loop and use to take the organism (Q).

-         Streak the wire loop containing the organism (Q) on the petri dish (3 lines).

-         Incubate the petri dish for 24-48 hours.

·        CITRATE TEST

-         Sterilize the wire loop and use to make a smear of organism (Q) on the citrate slant.

-         The inoculating needle should sterilized and used to stab the citrate slant.

-         Incubate for 24-48 hours.

·        GLUSCOSE & LACTOSE TEST

-         The wire loop should be sterilized and used to collect the organism (Q).

-         The wire loop should be dipped into the glucose broth and then shake the glucose broth

-         Incubate for 24-48 hours.

-         Repeat this step for lactose broth.

·        MR-VP TEST

-         Sterilize and use the wire loop to collect the organism (Q).

-         Dip the wire loop into the MR-VP medium and shake.

-         Incubate the MR-VP broth for 24-48 hours.

-         After 24-48 hours, the broth divide into two with one poured into the sterile test tube.

-         3 drops of Methyl Red should be added to one of the test tubes containing the broth and 40% Potassium Hydroxide + 16% α-naphthol to the second test tube containing the broth.

Record all your observations

·        TRIPLE SUGAR IRON AGAR (TSIA) TEST

-         The wire loop should be sterilized and used to collect the organism (Q).

-         Smear the organism on the TSIA slant.

-         Sterilize the inoculating needle and use it to stab on the TSIA slant.

-         Incubate for 24-48 hours.

·        INDOLE TEST

-         Sterilize the wire loop and used to pick the organism (Q)

-         Dip the wire loop into the indole agar broth and shake.

-         Incubate for 24-48 hours.

-         After 24-48 hours, Kovac’s reagent should be added to the broth and note your observations.

RESULTS/OBSERVATION

TESTS	EXPECTED OBSERVATIONS	RESULTS	INFERENCE
Oxida se Test	If the filter paper becomes colourless after a few Seconds.	-	Organism Q can’t produce  Cytochrome c Oxidase capable  of reducing  oxygen.
Citrate Test	If colour change from green to blue.	+	Organism Q can  utilize the citrate.
TSIA  Test H2S Slant Butt Gas	If black colouration is observed If pink colouration observed if yellow colouration observed if there’s breakage of agar	+      B      A      +	H2S is present Presence of base Presence of acid Gas is Present (Only glucose is fermented)
Indole Test	If on addition of Kovac’s reagent, there was no  formation of red ring.	-	The organism do  not have  the  ability to  hydrolyze amino acid Tryptophan  to indole
Motili ty Test	If the organism diffused to other parts of the agar	+	Organism Q is  motile
Catala se Test	If on addition of Organism Q to Hydrogen peroxide, bubbles were observed.	+	Organism Q was able  to detoxify  Hydrogen Peroxide.
Methyl Red Test	If on addition of methyl Red, colour changed to red.	+	Organism Q has  the ability to produce  acid.
VP Test	If after 30 minutes on addition of 40% KOH and 16% α-Naphthol, the colour remained unchanged.	-	Organism Q can’t  split glucose to  acetoin and can’t ferment butane-diol.
Starch Hydrolysis Test	If on addition of gram’s iodine, a    clear zone was noticed  around the streaked zone.	+	Organism Q is  able to utilize  starch.
Glucos e Test	If colour change from purple to yellow and there was gas collection inside Durham tube	+	Glucose sugar  was fermented  with the  production of   acid.
Lactose Test	If no colour change and absence of gas collection in Durham tube	-	Organism Q  cannot ferment Lactose  sugar.

DISCUSSION

If the above result is true for organism Q then the organism could be identified as Salmonella typhimurium.  It is a gram-negative organism that has a rod-like shape. It could be identified by a series of biochemical tests carried out on the unknown organism Q. The tests include motility, methyl red, lactose, VP, citrate test, etc.

          The major reason for the differences between actual and theoretical result is that during the various test conducted, some errors are bound to occur, these error tends to alter the results a little while theoretical results were obtained after series of experiment.

        In determination of unknown organism Q which was identified to be Salmonella typhimurium, lots of tests were done but the most pronounced in identifying the organism are glucose test, citrate, Methy Red, lactose and TSIA.

ECOLOGY

Salmonella typhimurium are part of bacteria flora normally found in man and animals (e.g reptiles, bird, wild and domestic animals) and are widely distributed in the natural environment e.g in water contaminated with faeces but growth is less in clean water and survival is also low. Salmonella typhimurium can also be found in wide range of sources in unfavourable environmental condition.

Survival rate:

-         87 days in tap water

-         115 days in pond water

-         120 days in pasture soil

-         280 days in garden soil

-         Over 30 months on dried bovine manure

-         28 months in avian faeces

PATHOGENICITY

This is the ability of an organism to cause disease. Salmonellosis (Infection caused by Salmonella) is a major environmental health problem. The true level of Salmonellosis is difficult to access accurately. The rate of disease causing of Salmonella typhimurium is very high. It is disease that affects reportedly about 2 million Americans each year and is common throughout the world. Salmonellosis is a worldwide disease of man and animal and spread from person to person. Gastro-enteristis is the major clinical manifestation of salmonellosis. Salmonella typhimurium usually invades the surface of intestine in humans causing inflammation that is more severe in the cecum with less inflammation in the colon and Tuttle or no inflammatory change in the ileum.

DESCRIPTION OF ORGANISM Q (Salmonella typhimurium)

      This bacterium causes typhoid fever and has a rod shaped conformation and is aerobic (they require oxygen to survive), and it’s also gram-negative (meaning it has three (3) layers of cell membrane). First outer membrane; the thin peptidoglycan layer gives its characteristics of gram-negative. It is a motile bacterium due to possession of flagella. It infects the small intestine, liver, spleen and bloodstream of human. Salmonella typhimurium is contacted through ingestion of contaminated food or water and its classification is as follows;

KINGDOM                                                    Bacteria

PHYLUM                                                                   Proteobacteria

CLASS                                                                        Gammaproteobacteria

ORDER                                                                       Enterobacteriales

FAMILY                                                                      Enterobacteriaceae

GENUS                                                                       Salmonella

SPECIE                                                                       typhimurium

CONCLUSION

       Identification of an organism can be achieved by carrying out various biochemical tests and it is very vital in order to treat a patient effectively.