Microbiology Unknown Lab Report

Madelyn Barr

October 11, 2017

Unknown I Lab

Unknown #30

Unknown I Lab Report

Abstract

        The “Unknown I Lab” was performed in order to evaluate the student’s ability to identify an unknown organism based on a series of tests and observations. Accurately identifying microorganisms is important for a variety of reasons. One of the most medically relevant reasons is being able to determine the causative agent of a patient’s illness so that you can treat it properly. I performed four tests in order to confidently classify my unknown organism. I began with a blood agar hemolysis reaction and then utilized single colonies from that plate to perform a Gram stain, catalase test, and a bile esculin test. A gamma-hemolytic blood result, positive Gram stain with cocci in chains result, negative catalase test, and positive bile esculin test led me to believe that unknown #30 is Enterococcus faecalis.

Introduction

        The purpose of this lab was to determine the identity of my unknown organism. I had unknown #30. The possible organisms available were Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Enteroccus faecalis, Streptooccus pneumoniae, and Streptococcus thermophiles. Staphylococcus aureus is present in most environments frequented by humans and is also readily isolated from fomites. Predispositions to infection include poor hygiene and nutrition, tissue injury, preexisting primary infection, diabetes, and immunodeficiency. S. aureus can cause localized cutaneous, systemic, and toxigenic infections. Localized cutaneous infections include folliculitis, furuncles, carbuncles, and impetigo. Osteomyelitis and bacteremia comprise the possible systemic infections. Toxigenic diseases include food intoxication, Staphylococcal scalded skin syndrome, and toxic shock syndrome. Staphylococcus epidermidis often lives on skin and mucous membranes of the human body. It is often the causative agent of endocarditis, bacteremia, and urinary tract infections (UTIs). Streptococcus pyogenes is the most serious streptococcus pathogen. Humans are its only reservoir, where it inhabits the throat and nasopharynx. S. pyogenes can enter through the skin and pharynx by way of contact, droplets, food, or fomites. Children are the predominant group affected by this organism. The most common diseases among children are impetigo and streptococcal pharyngitis. If left untreated, scarlet and rheumatic fever, septicemia, pneumonia, streptococcal TSS, and acute glomerulonephritis can occur. Enterococcus faecalis, otherwise known as Group D Streptococcus, is a normal colonist of the human intestine. It can cause opportunistic urinary, wound, and skin infections. Immunodeficient individuals are often susceptible to infection. Infections by Streptococcus pneumoniae usually occur when bacteria cells are aspirated into the lungs. Pneumococci multiply and induce an overwhelming inflammatory response. S. pneumoniae causes diseases like pneumonia and otitis media. Streptococcus thermophiles often inhabits the oral cavity, skin, nose, and genital tract. It is not very invasive, so dental and surgical procedures facilitate entrance. It causes a variety of diseases such as bacteremia, meningitis, abdominal infections, and tooth diseases. The most serious one is subacute endocarditis. Because S. thermophiles can colonize the heart by biofilm production, people with preexisting heart disease are at high risk for infection. There were multiple tests available to identify which organism I had. These included blood agar hemolytic reactions, Gram stain, catalase, coagulase, bacitracin and optochin susceptibility, bile esculin, bile solubility, and salt tolerance.

Materials and Methods

         At the beginning of this lab, I randomly selected an unknown broth labeled #30, which contained one organism. I was instructed to streak my broth onto a blood agar plate using a metal loop.  The plate was incubated overnight at 37°C and evaluated the following lab for a hemolytic reaction. Blood agar plates contain 5% sheep red blood cells and 95% trypticase soy agar (TSA). A complete lysis of the red blood cells indicates β-hemolysis, where a clear zone is observed around the organism’s growth. Hemolysins produced by the bacteria break down the red blood cells. Partial lysis is an α-hemolytic reaction. There is a breakdown of hemoglobin to met-hemoglobin, which is then reduced and causes a greening of the blood around the colonies. No lysis of blood indicates γ-hemolysis, where there is growth but no clear zone nor green color. The next test I performed was a Gram stain. I began by selecting a single colony from the blood agar plate and smearing it onto a glass slide. After staining the smear, I observed my organism under the microscope. Purple cells indicate Gram positive organisms while red cells indicate Gram negatives. Gram stains also display the morphology ad arrangement of the cell. All of the available organisms were Gram positive and cocci-shaped. Staphylococci are arranged in grape-like clusters and streptococci are in chains. The third test I performed was a catalase test to differentiate between the staphylococci and streptococci families. This is done by picking up a single colony from a TSB plate, smearing it onto a glass slide, and dropping hydrogen peroxide onto the smear. The formation of gas bubbles is a positive result. No gas bubbles is negative. All staphs are catalase positive because they contain cytochromes in their electron transport systems, which contain catalase. Hydrogen peroxide is broken down by catalase into water and oxygen, producing gas bubbles. It was important that I plated my organism onto a TSB plate, allowed it to incubate at 37°C for 24 hours, and picked a colony from there instead of the blood agar plate because red blood cells contain cytochromes, which could lead to a false positive. My final test was a bile esculin test. A bile esculin slant contains peptone, beef extract, bile, esculin, ferric citrate, agar, and distilled water. This medium tests for organisms that grow in the presence of bile and hydrolyze esculin. A black complex is produced when the bacteria hydrolyze esculin to esculetin, which then reacts with ferric ions in the medium. Growth and esculin hydrolysis indicate positive, while negative yields no growth or black complex. This test is important for identification of Enterococcus faecalis. I fishtailed my organism onto a bile esculin slant and incubated it at 37°C for 24 hours.