Lab 3: Sputum Cultures

In lab this week we were given a case along with sputum cultures on sheep blood, chocolate and MacConkey agar.  My case stated that a 48 year old man who had a long history of alcoholism was admitted to the ICU with hypotension and GI bleeding.  He was intubated and remained ventilator depended for several weeks.  He developed fever and his chest radiograph showed an infiltrate and changes consistent with multiple small abscesses.   

I identified 2 isolates based on colony morphology.  The first isolate was greenish-gray with broken glass appearance and beta hemolytic on SBA and smelled like grapes.  On chocolate it formed large colonies with spreading margins.  On MacConkey it formed slight pink colonies meaning it is a non lactose fermenting gram negative rod.    The presumptive identity for isolate 1 was non lactose fermenting gram negative rod.   Based on the colony morphology I performed a oxidase test and spot indole test.  The oxidase was positive and the spot indole was negative confirming the isolate as Pseudomonas aeruginosa.

Pseudomonas aeruginosa on SBA

Pseudomonas aeruginosa on Mac

The second isolate was small, white and opaque on SBA and chocolate and did not grow on MacConkey. Because it didn’t grow on Mac I knew it was gram positive but I did a gram stain to determine the morphology.  The gram stain showed gram positive cocci in clusters so I presumptively identified the isolate as Staphylococcus.  A catalase test was positive and the staphaurex (coagulase) test was negative.  My final ID was coagulase negative Staphylococcus which is normal flora in the throat.



Gram stain showed gram positive cocci in clusters


 

Positive catalase test



P. aeruginosa is seen in hospital acquired aspiration pneumonia.  The patient was most likely immunocompromised because of his alcoholism and because he had been intubated for several weeks he most likely aspirated and inhaled gastric contents containing the bacteria into his lower respiratory tract. 

Week 3: Upper and Lower Respiratory Tract Infections

This week we discussed upper and lower respiratory tract infections.  When diagnosing infections of the respiratory tract, normal flora in the throat must be considered.  Coagulase negative Staphylococcus, diphtheroids, Viridans streptococcus, and Neisseria spp are the most common normal flora found in the throat.  The main cause of pharyngitis and tonsillitis is viruses however the when the cause is bacterial the etiologic agent is usually Streptococcus pyogenes.  If S. pyogenes is suspected there is a rapid direct antigen test that can be done.  The first step is extraction of the antigen with enzymes.  The antigen in solution if present then reacts with the antibody to give a positive result.  If positive the report should say “positive for Group A Streptococcus antigen” and the patient should be treated.  If negative the report should say “negative for Group A Streptococcus antigen” and the specimen should be cultured.  Most of these rapid tests are chromatographic immunoassays. 

Lower respiratory tract infections include bronchitis, bronchiolitis, and pneumonia.  Possible pathogens include S. pneumonia, S. aureus, Klebsiella pneumonia, p. aeruginosa, and H. influenza.  Definite pathogens are M. tuberculosis, M. pneumonia, and Legionella. Legionella is primarily found in water and is transmitted by inhalation of infectious aerosols.  It was first isolated after an America Legion Convention in which a large number of people began to show nonspecific symptoms including fever, shills, cough, myalgia, headache, chest pain, and diarrhea.  Pneumonia and other organ involvement can also be seen.  Infection with Legionella was given the name Legionairre’s Disease.  Pontiac fever is similar to Legionairre’s disease however there is no pneumonia and no organ involvement. 

Here is a picture that shows how Legionella pneumophila replicates inside a host phagocyte. 

Lab 2: CNS infections and CSF

This week in lab we continued our focus of sterile fluids by examining cerebral spinal fluid.  Were all given a case study and a CSF specimen in order to determine what type of infection was present.  My case involved a 65 year old man who was admitted to the hospital in a coma.  His symptoms included a fever and stiffness in the neck.  He did not respond to verbal commands but did respond to painful stimuli to his leg. 

My first step was to perform a gram stain.  After examining the slide I gave the specimen a quantitation grade of 4+ meaning there were more than 30 per oil immersion field.   The gram stain showed gram positive rods forming palisades. Some rods formed short chains and pairs in the form of “L” and “V” letters. 

Gram stain of CSF specimen

I then examined the SBA and chocolate agar plates for growth.  Both showed small, translucent gray colonies.  The colonies that grew on sheep blood agar showed a small zone of beta hemolysis.



Colonies on SBA




The same blood agar plate examined with transmitted light allows you to see the small zone of beta hemolysis.

Based on the gram stain and colony morphology my preliminary ID was possible Listeria.  I then performed several direct tests to confirm the ID.  The catalase, bile esculin, and CAMP tests were all positive.  Growth in motility test medium showed growth away from the stab line in an “umbrella” shaped.  The crystal GP confirmed the isolate was Listeria monocytogenes. 

If we refer back to the case study, the elderly man had a fever which is indicative of infection and stiffness in the neck is the most recognizable symptom of a CNS infection.  His symptoms correlate with Listeria monocytogenes which is one of the bacterial agents that commonly causes meningoencephalitis. 

Week 2: Urinary Tract Infections

Urinary tract infections are the most common bacterial infection and can affect people of all ages and both sexes. UTIs are more common in females and in women of child-bearing age. UTIs are also the most common infectious complication in pregnant women. Types of UTIs include asymptomatic, pyelonephritis, urethritis, cystitis, and acute urethral syndrome.

Click on the following link for more information on the types of urinary tract infections, symptoms and treatment options.
More info on UTIs

Urine collection should be done in the early morning in a sterile screw capped cup or a tube containing preservative. There are several different methods that can be used to collect the specimen. A clean catch midstream (CCMS) is the most common method used because it is easy to collect and noninvasive. However contamination can occur unless collected properly. Straight catherization can be used to collect the urine with less contamination however it is more invasive and can introduce bacteria to the patient and lead to nosocomial infections. Collection from an indwelling catheter is a noninvasive method but has a high risk of contamination of the specimen. Colleting urine via suprapubic aspiration is the most reliable method because there is no contamination and it can also be used to culture for anaerobes. However this is a highly invasive procedure and can have associated risks.

After collection rapid screening tests can be done such as a gram stain, detection of pyuria, chemical tests (nitrate reduction and leukocyte esterase production), and colorimetry. If the screening tests give positive results for bacteria, a urine culture should be done and colony count should be conducted for each isolate to determine the significance. Direct tests can then be used to determine the identity of the organisms.

 

Lab 1: Blood Cultures

This week in lab we were all given a case study along with a blood culture bottle in order to determine what type of infection was present.  My case was based on a 25 year old woman who was admitted to the hospital for treatment of acute leukemia.  On admission she presented with fever and pain originating from a gingival lesion.  Blood cultures were negative on admission.  She started chemotherapy and developed neutropenia and on day 7 of aplasia, she developed fever associated with gingival pain and pus at the entry site of the central line. 

My first course of action was to make a gram stain of the blood culture. (shown below)

Gram Stain of specimen

Because the gram stain showed gram positive cocci in clusters I plated the specimen on SBA and incubated it overnight at 35°C.  The next day I examined the plate and found small, creamy white, opaque, colonies. (shown below)



 Growth on Sheep Blood Agar



Based on the gram stain and colony morphology my preliminary ID was Staphylococcus epidermidis.  I then performed a catalase test and a staphaurex test.  The catalase test was positive confirming the Staphylococcus genus.  The staphaurex test was negative as well as the tube coagulase test which rules out S. aureus.  Trehalose and mannitol fermentation tests were also performed and were both negative which further confirms the preliminary ID.  A crystal GP was done to confirm S. epidermidis.

S. epidermidis is normal flora on the skin and is common in infections with prosthetic devices such as catheters and shunts.  The oral mucosa is also considered to be a portal of entry.  The bacteria most likely infected her oral mucosa which caused the gingival pain.  After the chemotherapy compromised her immune system the bacteria spread into the blood causing sepsis. 

Week 1: Septic Shock

This week in lecture we discussed sepsis and septic shock. Sepsis is a systemic inflammatory response syndrome plus a documented infection.  Septic shock is sepsis with hypotension despite fluid resuscitation, and evidence of inadequate tissue perfusion.  Bacteria can either originate within the cardiovascular system from endocarditis, mycotic aneurism, or from an IV catheter or the bacteria can enter the blood through the lymphatic system from another site of infection such as a urinary or respiratory tract infection.  Some common organisms that cause intravascular bacteremia are S. aureus, S. epidermidis, S. pneumoniae, HACEK organisms, and viridians streptococci.  Extravascular bacteremia can be caused by E. coli, K. pneumonia, P. aeruginosa, S. aureus, and S. pneumoniae. 

If sepsis is a concern the physician will order blood cultures to be taken and examined by the laboratory.  A blood culture is blood obtained form 1 venipuncture site regardless of the number of bottles filled.  Bottles are held for 5 days unless they are determined positive by the automated system.  The BacT/ALERT is an automate system which detects a color change of the pH sensor on the bottom of the bottles.  When microorganisms from in the bottle CO₂ is released and generates free hydrogen ions causing a decrease in pH. The pH change results in the sensor color changing from green to yellow. 

Technologists must be aware of common contaminants that can be found in blood cultures.  S. epidermidis, Corynebacterium sp, Micrococcus, Viridans strep, and Bacillus sp are a few examples of contaminants.  I would like to know more about how technologists confirm it is a contaminant and if there is any additional testing that should be done. 

I’ve also posted an article is that I found interesting that was written after Jim Henson died of septic shock from S. pneumoniae. It mentions how time sensitive these infections can be and without immediate treatment the infection can be fatal.  (Click on the title of this post for the link)

About Me

Hi! My name is Laura and I am a graduate student in the Clinical Lab Science program at UAB.  I graduated from Auburn University with a degree in Microbiology and a minor in Spanish.  After teaching science to eleventh and twelfth graders for a year I decided to go back to school to get my masters.  This summer I am enrolled in an Infectious Disease course where I will be learning about different diseases as well as the proper testing techniques that should be used in a clinical laboratory.  Each week I will be posting something interesting that I learned that week during the lecture session as well as the lab.  Please feel free to comment or ask questions! Enjoy!