Introduction
Gram staining was developed by Christian Gram in the 1800’s, a Danish bacteriologist. (Smith and Hussey, 2005) It was the first differential staining technique and most common used in microbiology. Furthermore, bacteria are transparent and cannot be seen through the microscope. For that reason, Gram staining is an important tool for distinguishing between two main types of bacteria Gram-positive and Gram-negative. The Gram stain differentiates the Gram positive and gram-negative on the basis of their cell wall structure.(Menard, et al.,20150) Most bacteria gram positive or gram negative but they are a few gram variable bacteria and very small bacteria without a cell wall that do not have a gram reaction. For the purpose of this lab we are focusing on the two main types Gram + and Gram- bacteria. Bacteria are prokaryotes that have a cell wall; they are classified as bacillus (rod-shaped), Coccus (spherical) and Spirillum (Spiral) (Menard, et al., 2015). Therefore, in this lab we will examine be the
After using the gram Stain Techniques the gram positive bacteria turned purple and the gram negative pink showing the difference in each bacteria characteristics. Then, observing the morphological characteristics of bacteria, under a brightfield microscope, of the Citrobacter freundii a gram positive bacteria and the Staphylococcus aureus a gram negative bacteria.
We also learned what can go wrong when using gram staining that could produce errors in the testing. Hence, witnessing the different results that occurred when too much decolorizer is added or heat fixing the glass slide incorrectly. Learning the gram staining techniques have helped us understand how bacteria can be identify for proper treatment and for future studies of bacterial
I identified the genus and species of an unknown bacterial culture, #16, and I applied the following knowledge of morphologic, cultural and metabolic characteristics of the unknown microorganism according to the laboratory manual as well as my class notes and power point print outs. I was given an incubated agar slant labeled #16 and a rack of different tests to either examine or perform myself; the tests are as follows: Gram Stain; Nutrient Gelatin Test; Carbohydrate Fermentation; Dextrose, Lactose and Sucrose; IMVIC tests; Citrate, Indole, Mythel-Red and Vogues Proskauer test; as well as a Urease and TSI Test.
One bacterium was gram negative. It underwent four different tests. These tests were the EMB test (Eosin Mehylene Blue), the Sulfur Indole Motility (SIM) test, the Urease test, and the Simmon’s Citrate Utilization test. The EMB test checks for a bacteria’s ability to ferment lactose. This test is accomplished by placing the bacteria on Eosin Methylene Blue agar. The agar is selective for gram negative bacteria and those bacteria that can ferment lactose will have colored growth, usually a metallic green sheen.
The purpose of this study is to identify an unknown bacterium from a mixed culture, by conducting different biochemical tests. Bacteria are an integral part of our ecosystem. They can be found anywhere and identifying them becomes crucial to understanding their characteristics and their effects on other living things, especially humans. Biochemical testing helps us identify the microorganism present with great accuracy. The tests used in this experiment are rudimentary but are fundamental starting points for tests used in medical labs and helps students attain a better understanding of how tests are conducted in a real lab setting. The first step in this process is to use gram-staining technique to narrow down the unknown bacteria into one of the two big domains; gram-negative and gram-positive. Once the gram type is identified, biochemical tests are conducted to narrow down the specific bacterial species. These biochemical tests are process of elimination that relies on the bacteria’s ability to breakdown certain kinds of food sources, their respiratory abilities and other biochemical conditions found in nature.
After the end of the experiment the unknown 10 sample was Staphylococcus epidermidis. Came to this conclusion by first beginning with a Gram Stain test. By doing this test it would be easier to determine which route to take on the man made flow chart. Gram positive and gram negative bacteria have a set of different tests to help determine the unknown bacterium. Based on the different tests that were conducted in lab during the semester it was determined that the blood agar, MSA, and catalase test are used for gram positive bacteria while Macconkey, EMB, TSI, and citrate tests are used for gram negative bacteria. The results of the gram stain test were cocci and purple. This indicated that the unknown bacteria were gram positive. The gram stain test eliminated Escherichia coli, Klebsiella pneumonia, Salmonella enterica, and Yersinia enterocolitica as choices because these bacteria are gram negative. Next a Blood Agar plate was used because in order to do a MSA or a Catalase test there needs to be a colony of the bacteria. The result of the Blood Agar plate was nonhemolytic. This indicated that there was no lysis of red blood cells. By looking at the plate there was no change in the medium. Next an MSA test was done and the results showed that there was growth but no color change. This illustrates that the unkown bacteria could tolerate high salt concentration but not ferment mannitol. The MSA plate eliminated Streptococcus pneumonia and Streptococcus pyogenes as choices since the bacteria can’t grow in high salt concentration. Staphylococcus aureus could be eliminated because not only did the unknown bacteria grow but also it didn’t change color to yellow. Lastly a Catalase test was done by taking a colony from the Blood Agar plate...
I began my test to classify my unknown bacteria by performing the Gram staining because according to the first period procedure of the laboratory manual and the Appending H, it was the first test that should be done to plan and proceed to the next tests. Washed bottle of distilled water, three slides, and Gram-staining reagents
The purpose of this investigation was to identify an unknown bacterium. “At any time there are millions of bacteria living around, on, or inside us” (The Plague). Bacterium can’t be identified by merely looking at it. Many bacteria have the similar appearances in growth. “In most cases, detection is based on the reaction of an enzyme with a certain substrate” (Sigma-Aldrich). Identification is usually based on the results of the bacterium’s cells metabolic capacities.
The objective of this lab was to identify unknown bacteria culture by using various differential tests. There are many reasons for knowing the identity of microorganisms including to find the correct antibiotic to treat infections the bacteria may have caused. All the methods and techniques used to identify unknown bacterium #79 was learned in the microbiology laboratory.
Each test that was used in the lab for the unknown bacteria had been performed on many different bacteria and shown that each test has different results depending on the bacteria given. The first test, the Gram stain, confirmed that the unknown bacterium was a gram negative bacilli. After performing the remainder of the tests and comparing them to the twelve negative bacteria that it could be out of it was basically a process of elimination. Basically looking at all the results and seeing which tests separated positive verses negative results the most. After reviewing all of the tests the first test that stuck out besides the gram stain was the lactose fermentation, followed by the citrate utilization test and then by the indole test. The lactose fermentation test eliminated seven of the 12 bacteria. From the five bacteria left the citrate utilization test eliminated who more of the bacteria, and last the indole test eliminated two of the three bacteria left leaving only one bacterium left. After comparing the results to the results of the 12 tests and separating which tests were positive and negative for each it was obvious that the bacteria had to be Shigella
Coli. The most prominent growth was seen on the plate with Luria-Bertoni (LB) broth and ampicillin (amp) treated with +pGLO, as there were four separate and defined bacterial colonies with surrounding satellite colonies, each of which contained upwards of 20 cells visible to the human eye. Similarly, the plate with LB, amp, and arabinose (ara) treated with +pGLO showed three defined colonies, but no satellite growths. However, when the UV light was used to examine the bacterial colonies of that plate, they gave off a green fluorescence absent from other plates. Comparatively, the LB plate treated with +pGLO showed approximately equal growth to both the LB/amp/ara, and LB/amp plates. However, when examining the -pGLO plates, it was clear that there was conclusively no growth nor was there any fluorescence when the UV light was shown on the cells on either of the plates containing ampicillin or arabinose. However, the LB plate did show immense growth, such that the cells formed a bacterial lawn, yet the cells were not fluorescent under UV
After incubation, we checked the broth for the production gas and a color change from red to yellow which would indicate the presence of an acid. In order to perform a gram stain, we obtained a colony that we had grown on the TSA slant. Then we gathered one test tube containing tryptone broth, one containing citrate slant, and the last two containing MVRP broths. For the MVRP broths, one test tube should be labeled with MR and the next tube should be labeled VP. Next, we inoculated each test tube with the coliforms that we had confirmed as containing negative
It was then determined that unknown bacteria that could be categorized based on the mentioned gram reaction and morphology were either Cornybacterium xerosis or Bacillus subtilis; hence, a starch hydrolysis test, will be used to determine which bacterium could be the unknown. Metabolic test that are suitable for Gram positive cocci bacterium including, Micrococcus luteus, Staphylococcus aureus and Streptococcus faecalis were also included in the constructed dichotomous key; however, metabolic testing on GM+ cocci was not utilized due to gram staining
Coli. Each culture was grown in an M9 medium. One culture utilized glucose as a carbon source, while the other utilized succinate as a carbon source. Two other treatments of E. Coli were also tested, one without succinate and one without glucose. These two treatments were added as a baseline to compare how much succinate and how much glucose actually helped the E. coli grow. The two treatments were covered with parafilm and for the purposes of this experiment, will be called blanks. These cultures remained within their assigned group all day to measure the growth of E. Coli. The following process was repeated by all groups throughout the day. A cuvette was labeled with the sample that was being tested. The writing was at the top of the cuvette to prevent light from being disturbed and affecting results. 3 mL of the tested sample were placed in a flask using a sterilized 1 mL pipet. The spectrophotometer was then rezeroed with the corresponding blank inside. This was so that only growth would be measured. After recording measurements the flasks were returned to the incubator and the pipets were disposed of in a red biohazard bag. The contents of the cuvette were poured into 50% bleach to kill any E. coli. The cuvette was rinsed with distilled water. This process was repeated every 30 minutes over the course of eight and a half hours. Measurements at 12:00, 12:30, and 15:30 were missed due
The purpose of the study was to identify what are unknown bacteria by applying all the methods that we have learn in microbiology for the identification of are unknown. We apply the different test and be able to recognize the different characteristic of are unknown. Each test has its own purpose to help identify the bacteria by the reaction.
Phenotypic methods of classifying microorganisms describe the diversity of bacterial species by naming and grouping organisms based on similarities. The differences between Bacteria, Archaea and Eukaryotes are basic. Bacteria can function and reproduce as single cells but often combine into multicellular colonies. Bacteria are also surrounded by a cell wall. Archaea differ from bacteria in their genetics and biochemistry. Their cell membranes are made with different material than bacteria. Just like bacteria, archaea are also single cell and are surrounded by a cell wall. Eukaryotes, unlike bacteria and archaea, contain a nucleus. And like bacteria and archaea, eukaryotes have a cell wall. The Gram stain is a system used to characterize bacteria based on the structural characteristics of their cell walls. A Gram-positive cell will stain purple if cell walls are thick and a Gram-negative cell wall appears pink. Most bacteria can be classified as belonging to one of four groups (Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci, and Gram-negative bacilli) (Phenotypic analysis. (n.d.).
Leboffe, M. J., & Pierce, B. E. (2010). Microbiology: Laboratory Theory and Application, Third Edition 3rd Edition (3rd Ed.). Morton Publishing