The Lab Chronicles: My Experience in the Clinical Microbiology Lab – Weeks 5, 6 and 7

In this blog, I have combined my final three weeks in Microbiology. These last weeks mostly consisted of myself working on practice exams, as well as my final practical and theory exam. Additionally, I began working on my Clinical Project and started the testing process. However, I will highlight a few items performed over these last few weeks that have not been previously mentioned.

In preparation for my validation project of the new plastic blood culture bottles, I decided to get some practice in drawing blood cultures using a butterfly needle. The collection process for blood cultures is fairly straightforward: first, the outer seal and cap of the blood culture bottle is removed and the stopper is wiped with alcohol for disinfection. While the alcohol is drying, the vein of the patient is palpated (which means using your finger to find a vein to collect from). After a good vein is found, the skin of the patient is disinfected using alcohol. Then, the butterfly needle is inserted into the vein and the top of the blood culture bottle is pressed down on the tube holder for the blood to enter the bottle. The fill volume of blood culture bottles vary, but typically adult patients must have 8-10 mL of blood drawn for each bottle. However, this volume is smaller for children, as little as 0.5-2 mL of blood. Usually, for the collection of blood cultures, multiple sets (one set being an aerobic and anaerobic bottle) are collected. These sets must be collected from different sites and are used to detect contamination from normal skin flora.

Blood culture collection

Blood culture collection

During these final weeks, I also set up ESBL plates. ESBL stands for Extended Spectrum Beta-Lactams. This group of antibiotic resistant bacteria consists of Escherichia coli, Klebsiella pneumoniae and Proteus species that have undergone a point mutation that causes them to produce the enzyme β-lactamase. The initial identification of these antibiotic resistant bacteria is seen whenever they demonstrate resistance in susceptibility testing to any third generation cephalosporin or aztreonam. ESBL organisms can fall under certain categories based on their testing patterns. For example, ESBL Class A organisms are inhibited by clavulanic acid and demonstrate an increased zone of inhibition when an antibiotic is combined with it. The Class A ESBL mode of resistance is caused by a common β-lactamase enzyme present in Gram negative bacteria, known as the TEM-1 type β-lactamase. However, ESBL Class A can also be caused by the SHV-1 type β-lactamase. Class C ESBL organisms also exist that are not inhibited by clavulanic acid. This class of ESBL is caused by a mutation in the AmpC gene. Even though ESBLs are resistant to multiple antibiotics, there is no risk for infection control as they are not as easily transmissible as other common multiple resistant organisms. For this reason, ESBL screening is rarely performed in hospitals. The only time these organisms would be identified is within a clinical specimen with the antibiotic susceptibility results flagging for a possible ESBL. For those patients who have an identified ESBL, the only available group of antibiotics that can be given to treat their infection are carbepenems. Unfortunately, organisms resistant to carbapenem antibiotics have emerged, named Carbapenemase-producing Enterobacteriaceae (CPE).

ESBL Class A, demonstrating increased zone sizes when antibiotics are combined with clavulanic acid (CA)

ESBL Class A, demonstrating increased zone sizes when antibiotics are combined with clavulanic acid (CA)

On the final day in Microbiology, I presented an introduction of my Clinical Project to the laboratory staff. During this presentation, I explained the current testing procedure for blood cultures, describing the amount of time it takes to get a presumptive and final identification of a blood culture pathogen. I also explained the validation process and how it was two-fold in the transport of the pneumatic tube system, in addition to the preparation of smudge plates. Smudge plates, for those who may not know, are made from a concentration of the bacteria in the blood. 3 mL of a positive blood culture is transferred to a Serum Separator Tube and centrifuged. After centrifugation has occurred, bacteria lie on top of the separator. A drop is taken from directly above the separator and inoculated on Chocolate agar, streaking the drop in three different directions. In describing the methods of the validation, I explained how I would use one set of bottles for the reference method, which would be transported to the laboratory by walking. Additionally, another set would be used as the test method and transported through the PTS. A fifth bottle would also be drawn to use as a sterility control to determine if contamination was present within the venipuncture process. Once blood cultures came up as positive, I would inoculate two different types of plates. One plate would be streaked for isolation as in the regular procedure. The second plate would be the smudge plate.  From the regularly incubated plates, 4 hour and 24 hour identifications would be performed on the Vitek MS, and this mirrors the current procedure for positive blood cultures. For the smudge plates, I would be performing 2 hour and 4 hour identifications on the Vitek MS. In my presentation, I also stressed how travel and identification time could be improved by using both the PTS and smudge plates, respectively. In one quote, it was stated that for each hour of delay in treatment to septic patients, their survival rate decreases by approximately 8%. This quote further demonstrates the importance of my validation project and how the use of the PTS and smudge plates translates to improved patient care.

My presentation of my Clinical Project

My presentation of my Clinical Project

These past couple of weeks in Microbiology have been a great learning experience and I am so grateful to have had this insight into the clinical processes of the Microbiology laboratory! In my next rotation, I will be moving on to the Histology laboratory, and will be posting more blogs about my experiences there. Stay tuned!