11.1: Introduction

objectives

• Learn how to do some clinical tests (throat cultures and urine cultures) using selective and differential media.
• Learn about some significant anaerobic pathogens, and about how anaerobic bacteria are grown.
• Examine prepared slides of some vectors and the diseases that they transmit.

Health care providers will often be expected to obtain samples from patients to diagnose a particular infectious disease. When doing this, it is important to use aseptic technique and proper precautions to protect the person handling the sample, avoid contamination of the surrounding area, and preserve the integrity of the sample. Following proper aseptic technique guarantees that what grows on the Petri dish came from the patient’s sample and is not an environmental contaminant. There are numerous clinical tests that can be used to identify specific types of infections—you will use two of these in this laboratory activity.

Throat Cultures

health care providers will often take a throat culture from a patient complaining of a sore throat. This is most commonly used to diagnose strep throat, which is caused by Streptococcus pyogenes. Although there are now “rapid-strep” tests that may be used, a more traditional way of identifying this organism in a throat culture is to use Blood agar. This media contains 5% sheep’s blood, and allows for the distinction between types of hemolysis. Many bacterial species are α- hemolytic, which means they can partially break down red blood cells. Bacteria that are β- hemolytic can completely break down red blood cells; those that are γ-hemolytic do not break down red blood cells at all. Streptococcus pyogenes is a β-hemolytic organism— colonies of this species will completely lyse red blood cells, and so clear zones will be seen around the colonies on a blood agar plate (BAP). Other species, such as Streptococcus pneumoniae, are α- hemolytic, meaning that they can partially break down red blood cells—these colonies will have a greenish pigment around them on a BAP. Species that are non-hemolytic (γ-hemolytic) will grow on Blood agar, but no color change will be observed (e.g. Enterococcus faecalis, Streptococcus salivarius)

Note

For a review of hemolysis (including color images), see the Microbiology review site on Blackboard. The diagnosis of strep throat is important because if left untreated, it may progress to scarlet fever and/or a more serious infection that can result in cardiac tissue damage. A positive strep test indicates the need for antibiotic therapy.

Streptococcus pyogenes infections are not confined to the respiratory tract. This organism can also cause skin infections such as erysipelas, cellulitis and impetigo. In rare cases, some strains of S. pyogenes can invade and multiply in the fascia, causing necrotizing fasciitis, resulting in rapid tissue destruction with mortality rates that can exceed 40%.

Urine cultures

Urine cultures are taken to diagnose urinary tract infections (UTIs), a very common type of nosocomial infection (an infection acquired during a stay at a hospital or chronic care facility). Many types of bacteria can cause UTIs but E. coli is the most common culprit. Although urine within the body is generally microbe-free, some bacteria are picked up as urine leaves the body. Therefore UTIs are generally diagnosed by determining the amount of bacteria present—low numbers are not usually indicative of an infection unless the organism detected is a very serious pathogen that would not be part of the normal microbiota. To minimize the amount of bacteria from the external genitalia that are present in a urine sample, patients are usually asked to wipe the area before urinating, and a “clean catch” is taken by letting some urine go before collecting—this flushes away the external normal microbiota. A urine sample will often appear cloudy if an infection is present. Selective and differential media, like EMB and BA, can be used to determine the types of bacteria that are present in a urine samples. E. coli can be identified on EMB by the green metallic sheen of the colonies. Generally, more than 100,000/ml of one type of organism reflects significant bacteriuria (bacteria in the urine), but lower numbers of certain pathogens may also indicate infection. Types of UTIs include urethritis (infection of the urethra), cystitis (bladder infection) and pyelonephritis (kidney infection).

Anaerobic pathogens

Many bacterial species are aerobic or facultative (can grow with or without oxygen). A few pathogenic species are obligate anaerobes, which means they cannot grow in the presence of free oxygen. These pathogens would not cause superficial or lung infections, but rather may be responsible for infections within the body in environments where free oxygen is scarce. Infections occur by way of puncture wounds, ingestion, or when a body part is cut off from its blood supply via injury or disease.

One genus of pathogenic anaerobes is Clostridium. Members of this genus are gram positive, bacillus-shaped endospore formers. Pathogenic members of this genus include C. tetani, C. botulinum, C. difficile, and C. perfringens.

Note

Not all endospore formers are anaerobic—Bacillus anthracis, which causes anthrax, is an example of an aerobic pathogen that may cause infections in the lungs and on the skin.

Growing obligate anaerobes requires creating an environment that is free of oxygen. One way to do this is using the GasPak system. Agar plates are placed into a special jar and sealed tightly. A sachet is placed in the jar along with the plates—this sachet contains chemicals that bind up the free oxygen in the jar; an indicator strip is used to ensure that no free oxygen remains. In this lab you will be using the GasPak system to determine the oxygen requirements of two types of bacteria.

Vectors of pathogenic microbes

Disease-causing organisms can be transmitted in many different ways. Some are transmitted by contaminated food or water, some by respiratory droplet, and some by sexual contact. Some pathogens are also transmitted to humans by vectors including insects (flies, mosquitoes) and arthropods (ticks, mites). Vectors themselves are not microscopic organisms, but transmit microbes in one of two ways:

1. Mechanical vectors carry microbes on their body (e.g., a fly landing on your food).

2. Biological vectors carry microbes within their body, and will transmit to a new host via bite or sting (e.g., mosquito bite transmitting malaria; tick bite transmitting Lyme disease).

Diseases caused by vector-borne pathogens are difficult to control or eradicate because it would be necessary to kill all of the individual organisms that carry them. Disease prevention for vector-borne pathogens include the use of insecticides and insect repellents (which pose their own health risks), avoiding contact with the vector by using mosquito netting, or avoiding being outside in areas when/where the vector is most active. In this lab you will observe slides of a few common vectors, as well as the pathogens that some of them transmit.

Key Terms

obligate anaerobes, GasPak system, nosocomial infection, biological vector, mechanical vector, bacteriuria, α, β and γ hemolysis