Microbiology Labs I
- Page ID
- 3249
\( \newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} } \) \( \newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}} \)\(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\) \(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\)\(\newcommand{\AA}{\unicode[.8,0]{x212B}}\)
- 1: Media Preparation
- Bacteria and fungi are grown on or in microbiological media of various types. The medium that is used to culture the microorganism depends on the microorganism that one is trying to isolate or identify. Different nutrients may be added to the medium, making it higher in protein or in sugar. Various pH indicators are often added for differentiation of microbes based on their biochemical reactions. Other added ingredients may be growth factors, NaCl, and pH buffers.
- 6: Oxygen Requirements and Anaerobes
- An excellent way to determine the oxygen needs of your bacterium is to grow it in different oxygen environments---atmospheric oxygen of 22%, no oxygen at all (GasPak jar), and reduced oxygen at less than 10% (candle jar)--and compare the quality and quantity of growth.
- 7: Isolation of an Antibiotic Producer from Soil
- Soil is the major reservoir of microorganisms that produce antibiotics.Considering that soil is densely packed with microorganisms, it is not a wonder that many bacterial and fungal species have evolved over the eons to develop ways of inhibiting their neighbors for the benefit of their own growth. An antibiotic made by a microbe can inhibit many other soil microbes.
- 8: Bacterial Colony Morphology
- Bacteria grow on solid media as colonies. A colony is defined as a visible mass of microorganisms all originating from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike.
- 9: Kirby-Bauer (Antibiotic Sensitivity)
- The Kirby-Bauer test for antibiotic susceptibility (also called the disc diffusion test) is a standard that has been used for years. It has been superseded by automated tests, but the K-B is still used in some labs, or used with certain bacteria that automation does not work well with. This test is used to determine the resistance or sensitivity of aerobes or facultative anaerobes to specific chemicals, which can then be used by the clinician for treatment of patients with bacterial infections.
- 10: Antimicrobial Chemicals
- Many herbs and spices MAY have (some documented, some just hearsay) antimicrobial activity: thyme, cinnamon, garlic, ginger, chamomile, oregano, sage, echinacea, wasabi, etc. We are going to try some out in this lab: fresh or dried, leaf or powder. In addition, you will be running various antimicrobial chemicals against your bacteria. To save on time, for the identification of antibacterial spices, each group will extract a different plant tissue and prepare enough herb/spice extract for each gr
- 11: Bacterial Numbers
- Many studies require the quantitative determination of bacterial populations. The two most widely used methods for determining bacterial numbers are the standard, or viable, plate count method and spectrophotometric (turbidimetric) analysis. Although the two methods are somewhat similar in the results they yield, there are distinct differences.
- 12: Oral Biofilms
- Most of the time in the microbiology lab, we study free-floating bacteria in broths or bacteria in colony forms, and generally in pure culture. However, in the real world bacteria are usually interacting with other species in pretty sophisticated ecosystems. This assemblage of various organisms attached to a surface is called a biofilm , and the organisms that comprise it can include bacteria, plants, fungi, protozoa, and even multicellular animals, depending on where the biofilm is.
- 14: Use of the Microscope
- The microscope is absolutely essential to the microbiology lab: most microorganisms cannot be seen without the aid of a microscope, save some fungi. And, of course, there are some microbes which cannot be seen even with a microscope, unless it is an electron microscope, such as the viruses.
- 17: Gram Stain
- The gram stain, originally developed in 1884 by Christian Gram, is probably the most important procedure in all of microbiology. It has to be one of the most repeated procedures done in any lab. Gram was actually using dyes on human cells, and found that bacteria preferentially bind some dyes. The Gram stain is a differential stain.
- 21: Capsule Stain
- The capsule is a thick polysaccharide layer around the outside of the cell. It is nonionic, so the dyes that we commonly use will not bind to it. Two dyes, one acidic and one basic, are used to stain the background and the cell wall, respectively. The area between the 2 dyes is the halo of the capsule surrounding the cell. The capsule gives added protection to the bacteria, making it virtually impossible to be phagocytosed by white blood cells.
- 26: Catalase Test
- Hydrogen peroxide H₂O₂ is a by-product of respiration and is lethal if it accumulates in the cell. The enzyme catalase degrades the hydrogen peroxide in the cell before it can do any cell damage. It splits the H₂O₂ to free oxygen (bubbles) and water. Generally, the test reaction is very fast and obvious bubbles will be seen. Catalase is made by your own cells, as well as a variety of other cells, including many bacteria. This test is particularly important for the gram+ bacteria.
- 27: Oxidase Test
- The oxidase test is a key test to differentiate between the families of Pseudomonadaceae (ox +) and Enterobacteriaceae (ox -), and is useful for speciation and identification of many other bacteria, those that have to use oxygen as the final electron acceptor in aerobic respiration. The enzyme cytochrome oxidase is involved with the reduction of oxygen at the end of the electron transport chain.
- 28: Starch Hydrolysis
- The enzyme amylase is secreted out of the cells (an exoenzyme) into the surrounding media, catalyzing the breakdown of starch into smaller sugars which can then be absorbed by the cells for use. Iodine reacts with starch, producing a deep purple color. As starch is catabolized and converted to sugars, there will be less and less starch to react with the iodine.
- 29: Lipid Hydrolysis
- The enzyme lipase is excreted out of the cells (an exoenzyme) into the surrounding media, catalyzing the breakdown of tributyrin, a vegetable oil, into fatty acids which can then be taken up by the organism. This reaction causes the agar, normally a light blue opaque color to clear around the growth area.
- 30: Casein Hydrolysis
- The enzyme caseinase is secreted out of the cells into the surrounding media, catalyzing the breakdown of milk protein (casein) into small peptides and individual amino acids which are then taken up by the organism for energy use or as building material. The hydrolysis reaction causes the milk agar, normally the opacity of real milk, to clear around the growth area as the casein protein is converted into soluble and transparent end products.
- 31: Coagulase Test
- The enzyme coagulase, produced by a few of the Staphylococcus species, is a key feature of pathogenic Staph. The enzyme produces coagulation of blood, allowing the organism to "wall " its infection off from the host's protective mechanisms rather effectively. There are 2 methods: slide test and tube test. The tube test is more accurate, but the slide test is faster.
- 33: Deoxyribonuclease (DNAse) Test
- Deoxyribonuclease (DNAse) agar actually has DNA in it. Deoxyribonuclease is an exoenzyme excreted from the cell which will break the DNA down into smaller molecules.
- 34: Urea Hydrolysis
- Urea can be broken down with the help of the enzyme urease, producing the alkaline product of ammonia plus carbon dioxide. That causes the pH indicator phenol red to turn a beautiful shade of hot pink.
- 35: Carbohydrate Use
- You are going to see 2 different ways to run sugar tests: phenol red sugar broths and the sugar disc methods. Some of the sugars come in phenol red broth, already with sugar in it lactose, mannitol, glucose (dextrose), and sucrose.
- 36: Bile Esculin
- Bile esculin agar comes in a slant form. It is 2 tests in one---resistance to 40% bile AND the use of the sugar esculin. Bile is a very inhibitory chemical, particularly at that concentration. IF the bacterium grows, the next question is whether it uses the esculin. If your bacterium cannot grow in the presence of bile, the esculin reaction will have to be obtained a different way, using an esculin sugar disc.
- 37: IMViC tests
- These 4 IMViC tests - Indole, Methyl red, Voges-Proskauer, Citrate + and H2S (actually 6 tests if you include motility and H2S) - constitute, perhaps, the most critical tests used for identification of bacteria after the gram stain. The test results from these 6 tests should carry more weight than almost any other tests, certainly higher priority than sugar results since they are more stable reactions.
- 38: TTC Motility Agar
- SIM deeps are a multi-test medium comprising 3 tests: sulfide (H2S gas), indole production, and motility. You have already tested for motility via the hanging drop slide, and here is an additional way to determine it, with less muss and fuss. In fact, there is an even better way to determine motility, motility agar with tetrazolium dye, that you will also be using in lab.
- 39: Gelatin Hydrolysis
- Gelatin is a protein that is solid at room temperature. If the bacterium makes the enzyme gelatinase (which optimally is produced at 25º C, not 37º C), the gelatin is hydrolyzed and becomes a liquid. There is no indicator or reagent added, simply solidification or liquefaction.
- 40: Decarboxylation of Amino Acids
- There are 3 decarboxylase enzymes we can test for--arginine decarboxylase, ornithine decarboxylase, and lysine decarboxylase. These enzymes break the bond holding the carboxylic (-COOH) group to the rest of the amino acid. As a result, the end product is a basic chemical which causes the pH to go up, changing the indicator brom cresol purple to turn purple.
- 49: Bacteriophages (Experiment)
- Bacteriophages are viruses which infect bacteria. In this lab, 2 kinds of bacteriophages will be used---T4 and phi 174 viruses. Their host bacteria are 2 different strains of E. coli,so these bacteriophages are called coliphages. The purpose of using 2 different viruses is to show the specificity of a virus for its host, even for these little bacterial viruses. The liquefied tryptone soft agar, into which the bacteria and viruses are placed, has less agar concentration than normal liquefied agar
- 53: Serology - Antigen-Antibody Tests
- Any feature that can elicit an immune response is called an antigen. The immune system makes proteins called immunoglobulins or antibodies which bind to an antigen to either directly neutralize the antigen or cause it to be cleared more efficiently by other components of the immune system. Antibodies linked to large particles such as latex beads can be used to agglutinate microorganisms using specific reactive antibodies.