Gram StainWhen a group of cells is stained with Crystal Violet and with Iodine, Ethyl Alcohol is used to remove any color it can. A counter stain is done to help identify one type with Safranin. If the cell type is gram positive it will remain purple because the Ethyl Alcohol could not remove all of the Crystal Violet. If it is Gram- then the violet color would be gone and only a red counter stain would remain.G+=Purple Color G-=Red ColorGram -
Carbohydrate FermentationIn this test two types of broths were used. One was Phenol Red Lactose and Phenol Red Dextrose. In each tube, this is the only carbon source. When inoculated with a bacteria, there are multiple outcomes. If the bacteria can utilize it, it will grow. There is a Phenol Red pH indicator in the solution that will show if acid was produced in fermentation. A change in color from red to yellow shows this. Also, there is a Durham tube in the broth tube. This is a small tube, upside down, that will collect any gas that was produced from fermentation. +Fermentation: all + -Fermentation: < 2 +Lactose: Growth but no fermentation
Dextrose: Growth, produced acid, but no gas produced.
Negative for both dextrose and lactose fermentation
Colony GrowthEvery type of bacteria has its own characteristic growth. When a streak culture is done on a TSA pate and the plate is allowed to incubate, the bacteria will grow in a certain form.There is no specific outcome to look for. It is just to see a characteristic of this bacteria. The colony growth also doubled as a motility test. The unknown spread out over the entire agar plate covering it in a layer. Na Thioglycolate MediumA Fluid Thioglycollate Medium is used in this test. The unknown is inoculated through stab technique. This medium contains sodium which helps to bind free oxygen, which is toxic to anaerobes. This allows the anaerobes to grow in the medium too. There is also Resazurin, which is an indicator for oxidized environments. Depending on where the organism grows will show what type of environment this organism can live in. Anaerobe: Will only grow at bottom of tube. Facultative Anaerobe: Will grow throughout, but more abundant near top. Microaerophiles: Will grow right under pink layer in tube.
Aerobes: Will grow at the top of the tube.The unknown is a Facultative Anaerobe because it was tolerant to oxygen, but grew mostly in the anaerobic region. Indole TestThis test uses a medium that contains 1% tryptone in water. This will help see if the enzyme Tryptophanase is present in the bacteria. If it is, the Tryptophan components of tryptone will be broken down and converted to indole. This can be seen by adding Kovac’s reagent which will combine with the indole to form a red layer in the tube.+Tryptophanase: Kovac’s reagent combines with indole to make a red layer on the top surface. -Tryptophanase: There is no reaction with Kovac’sPositive for indole production, which means Tryptophanase is present. Methyl Red TestThis test is to see if the bacteria is capable of fermenting Dextrose. If capable there will be mixed acids produced by the bacteria from fermentation. Methyl Red is used to test the acidity of the solution. +Acid Fermentation: solution turns red in presence of Methyl Red. -Acid Fermentation: Solution will turn orange or yellow. Positive for acid fermentation from Dextrose usage. Voges-Proskauer TestThis test is used to determine whether or not the bacteria can perform a butanedio fermentation. This is found out by testing for acetyl methyl carbinol. This is a precursor to 2,3-butanediol. A solution called Barritt’s Reagent A and B are used to test for this chemical. When added the reagents will react with acetyl methyl carbinol turning the solution red.+ Butanediol fermentation: With Barritt’s Reagent A and B solution will turn red.
- Butanediol fermentation: