Microbiology Streak Plate Procedure

Question :

Describe the streak plate procedure. Make sure to answer in your own words in paragraph form (using complete sentences, do not copy from the lab manual). Be sure to include any necessary materials, and incubation procedures.

Answer :

The streak plate method is used isolate individual microbial cultures from a given sample. There are different types of streak plate method – quadrant streaking and two-phase streak. 

Quadrant streaking 

This is another type of streaking where there is intermittent heating in between. A loopful of culture was taken in and a pool of organism is made in one end of the petri dish. From this pool of microorganism simple zig-zag streak is done in one end of the petri dish without reaching the middle. Then the inoculation loop is incinerated. After incineration, the plate is now turned to 45 degrees and without taking the organism again the empty inoculation is streaked as if from the already streaked lines at; right angle. This procedure is repeated in the third side and fourth side as well. Quadrant streak is done when the concentration of the microbial colony is very high.  

Two-phase streak

Two phase streak is a technique where a single streak is diluted using another streaking technique at right angle with the first streak. The 

The organism A had a mucoid colony, with flat elevation, irregular in form, and undulated colonies. 

The colony morphology is circular, flat in elevation, and entire in margin. 

A loopfull of culture was pooled on a glass slide and heat fixed. The microbial film was smeared with crystal violet dye and left for 5 minutes. The excess dye was removed by passing it through running water. It was flooded with grams iodine that acts as the mordant, and left for 5 minutes. The slide was washed in running water. It was then decolorized using ethanol for 2 minutes and then washed with water. The slide was counter stained with saffranin and left for 5 minutes. The excess dye was removed by washing it in running tap water and observed under a microscope. Purple colored colonies are gram positive, while, pink colored colonies are gram negative organisms.  Apart from the cell wall characteristics, the gram staining is also used in identifying the shape of microorganisms, and the mode of arrangement of the organisms. 

• Organism A - Gram negative rod

• Organism B - Gram positive cocci 

The organism is a gram negative rod. The organism was appearing in a pink color taking the secondary stain saffranin. While visualizing under 1000x magnification, the cell was a clear rod shaped bacterium having saffranin, showing that it is a gram negative rod. 

The cell envelop is a gram negative one having minimal layers of peptidoglycan layers and an outer layer made up of lipo polysaccharides. As such the gram negative cell wall would be composed of peptideglycan layer surrounding the phospholipid bilayer following it with the lipo polysaccharide layer. This is the additional layer that the gram negative cells do contain. It is the presence of lipid in the outer layer that has aided in the removal of primary dye and mordant complex and in the fixing of secondary dye. 

Based on the colony morphology and the cell wall morphology the organism could be Escherichia coli. 

Use the results file to observe results you would have obtained on day 3 of the experiment. 

1. Detail the cell morphology and Gram reaction you observe for organism B. 

The organism is a coccus that is found in clusters. Based on the color in which it is observed in a gram staining it is found to be a gram positive organism. Considering both these it is said is a gram positive staphylococci based on its morphology. 

The cell wall is said to be a gram positive one. The gram positive cell wall is said to be lining the phospholipid bilayer in the cell. The gram positive cell wall is composed of more than 60 layers of peptidoglycan layer composed of N-acetyl muramic acid and N-acetyl glucosamine as its monomers interlinked with tetrapeptides. This multilayer peptidoglycan is what has locked the primary dye and mordant complex during the gram staining process.

Based the cell morphology and arrangement the organism could be Staphylococcus aureus. 

The test carried out was carbohydrate fermentation test specifically for lactose. The test was done by inoculating the given organism in a test tube containing lactose broth along with an inverted Durham’s tube. The tube would be incubated in at 37 degrees for 24 hours. When checked the next day, a positive fermentation would be intimated by the presence of gas produced in the Durham’s tube. The media would be supplemented with a pH indicator and when an acid end product is formed the color of the media changes to yellow color, showing that lactose is formed and acid end products are produced. 

The test carried out is the catalase test. Catalase test is used to check whether the organism is catalase positive or not. For this test a drop of hydrogen peroxide is taken on a glass slide, and a loopful of bacterial culture is added to it. If the organism produces catalase enzyme, we would see effervescence.  

Use the results file to observe results you would have obtained in this experiment. On day 5 of the experiment the first round of biochemical tests produced the results shown. Analyze the results for organism B. Answer the following questions in complete sentences in your own words.

1. Describe the results obtained for the first biochemical test used on organism B. Be sure to describe what you visualize do not just state + or - result. 

+ve for catalase.

The organism is positive for catalase test. This shows that the organism is producing catalase that has broken down the hydrogen peroxide solution to water and oxygen molecule. Catalase is an enzyme found normally on all aerobic organisms and protects them from toxic reactive oxygen species. 

The organism is aerobic in nature. The organism respires through aerobic mode and when it does produces reactive oxygen species during its metabolism, catalase enzyme breaks the reactive oxygen species and helps their cell from getting damaged. 

Use the results file to observe results you would have obtained in this experiment. On day 5 of the experiment the first round of biochemical tests produced the results shown. After analysis of this first round of biochemical tests could any organisms be ruled out for organism A? for organism B? Explain. Make sure to answer in complete sentences in your own words.

For organism A

The organism is a gram negative rod that is a lactose fermentor. 

We can rule out S. aureus, B. subtilis, S. lactis are gram positive organisms, hence ruled out. P. vulgaris is not a lactose fermentor and hence, it is also ruled out. 

Hence we are ruling out E. coli, P. vulgaris, and E. aerogenes as they are gram negative organisms. We are also ruling out B. subtilis as the organism observed is a cocci and not a rod. 

Use the results file to observe results you would have obtained in this experiment. The second round of biochemical tests ran on Day 5 of the procedure produced the results shown on Day 6 of the experiment.

Describe the procedures used for the second biochemical test ran on microbe A. Be sure to detail these procedures in paragraph form (using complete sentences, in your own words, and do not copy from the lab manual).

The test carried out is the Citrate utilization test. The test is mainly done to check whether the organism under study is capable of utilizing sodium citrate as the sole carbon source and ammonium salts (inorganic) as the sole nitrogen source. Simmons citrate agar is taken, autoclaved and slants were prepared in test tubes.  To the slant the given organism is inoculated and incubated at 37 degrees for 24 hours. The slants would be checked for a change in color. Growth in the medium along with change in the color is considered positive. 

The test carried out for the organism 2 is also the same test used for the first organism, which is the Citrate utilization test. The test is mainly done to check whether the organism under study is capable of utilizing sodium citrate as the sole carbon source and ammonium salts (inorganic) as the sole nitrogen source. Simmons citrate agar is taken, autoclaved and slants were prepared in test tubes.  To the slant the given organism is inoculated and incubated at 37 degrees for 24 hours. The slants would be checked for a change in color. Growth in the medium along with change in the color is considered positive.