10/13/2016
If you’ve ever had a sore throat swabbed to test for strep, you have experienced just one way bacteria cultures can be used to help sick patients get answers.
The Bacteriology Laboratory at Strong Memorial Hospital runs hundreds of tests around the clock to identify the bacteria and fungi that cause everything from urinary tract infections to food poisoning. This identification process is the first step in stopping sickness in its tracks and putting patients on the road to recovery.
There is a rainbow array of plates that medical technologists use to grow different bacteria. An ordering provider may suspect a certain type of infection and ask the lab to run a specific test, or tests, to confirm the identity of the culprit and see what drugs it best responds to.
Most bacteria are traditionally grown on an agar media plates that contain sheep’s blood. Historically, this has been the way to obtain bacterial growth in order to determine which organisms are “normal flora” and which bacteria are “bad,” causing disease or infection. Culturing allows “bad” bacteria to be tested for susceptibility to certain antibiotics. Advances in modern technology are now making this process more automated than ever before.
How it starts
If you have symptoms of a urinary tract infection, for example, your urine sample will be sent to the lab for testing from your doctor’s office. After it has been received, labeled, and entered into an electronic database for tracking, the specimen goes to a medical technologist.
He or she will then dip a tiny calibrated plastic loop into the urine and make a streak onto a sterile blood plate to start the process of growing bacteria. This process is called inoculation.
About 80 percent of all UTIs are caused by the bacteria E. coli, classified as Gram-negative bacilli due to the composition of the cell wall. The tech will also select media (another name for agar plates) that will only grow Gram-negative bacteria (called MacConkey agar in this case) to help identify the “bad” bacteria and prevent growth of other organisms.
“That’s what a lot of bacteriology is: knowing who the good guys are – the normal flora – and who the pathogens are,” said Jennifer Barrante, education coordinator for Microbiology at URMC. “We’re trying to enhance the recovery of our ‘bad’ guys.”
Since bacteria need warmth and time to grow, the inoculated plates are then placed into and incubator that looks like a refrigerator with a glass door, but warms up to body temperature instead of cooling. The plates are allowed to incubate for a minimum of 18 hours to give the bacteria time to grow.
After incubation, the technologist takes the culture and counts the number of bacterial “colonies” that have grown overnight. E. coli, for example, will appear as pink colonies on the MacConkey plate after it has incubated overnight. This is a quantitative culture because the quantity of bacteria reported is based on the actual number of colonies grown, multiplied by 1,000 to determine colonies per milliliter of urine.
Most cultures are not quantified this way. Instead of counting the number of colonies, cultures are usually read semi-quantitatively using a four-quadrant system.
Picture the circular plate evenly divided into four sections with a specimen streak in each quadrant. If growth appears only in the first quadrant where the specimen was inoculated, the reading is “one-plus.” If there is also growth in the second section of streaking, it’s 2+, and so on.
What else can you grow?
Lab techs are able to culture bacteria from a wide variety of biological material. This can be a swab of something as innocuous as a blister or a cut on your finger, to something as serious as spinal fluid to detect meningitis.
If a patient undergoes a biopsy on a tissue mass that’s suspected to be cancerous, part of the tissue specimen can be cultured to see if the growth is caused by an infection instead of cancer.
Blood specimens can also be cultured to detect bacteremia (bacteria in the blood) or sepsis (a severe reaction to an infection that may involve the blood) which can be caused by different organisms like staph, or Haemophilus.
Besides urine and blood, stool specimens are routinely cultured to identify bacteria – like Salmonella, Shigella, or Campylobacter – that cause food poisoning.
You can also test for fungal infections like ringworm, a yeast infection or oral thrush, which appears as white spots inside the mouth.
The lab also tests specimens for acid-fast bacilli, a group of bacteria that includes the cause of tuberculosis.
Automation
While these methods of culturing specimens have been around for many decades, new technologies allow labs to get the same results more quickly, and with less hands-on work.
There are several large instruments at URMC that perform testing on a molecular level. This kind of test is called a PCR, or polymerase chain reaction, and it uses small samples of DNA to detect patterns that indicate the presence of certain bacteria, viruses, or infections.
One PCR instrument can test stool samples for presence of C. difficile and Norovirus. That same instrument can use a nasal swab to test for MRSA. Another instrument allows for testing of the respiratory virus panel based on a nasal swab.
The newest instrument, the BD MAX, has replaced the traditional stool culture method for detecting Salmonella, Shigella and Campylobacter. It can also detect the stool parasites Giardia, Cryptosporidium, and E. histolytica when requested by a doctor.
This instrument reduces the time it takes the result to get to the doctor. A traditional stool culture can take 3-4 days to be resulted once received in the lab, but the BD MAX can get the same results within 24 hours.
Automated molecular instruments can also provide much faster results for some sexually transmitted diseases just hours after they are run on the instrument, rather than patients having to wait days for traditional cultures to yield a result.
Fighting back
Yes, PCR automation saves time, but identifying the causative agent of an illness is just one step in determining how it should be treated. Many specimens must still be cultured to determine their susceptibility – to find out which antibiotics will kill off the offending bacteria. This process is called susceptibility testing.
One method of S.T. is the Kirby-Bauer disk diffusion test. For this test, the surface of a large culture plate is swabbed with the pathogenic organism and small discs of antibiotics are placed on the plate. After incubating overnight, a tech will measure the diameter of the circles that have formed around each “pill” to determine what medications are most effective.
"We have different panels of drugs to test depending on the bacteria and specimen source the organism comes from in order to provide the best treatment options,” said Barrante.
She explained that using antibiotics incorrectly can wipe out populations of very susceptible bacteria and can leave more resistant strains behind. Or, organisms can swap resistance genes and cause “superbugs” that can’t be killed by antibiotics.
Susceptibility testing informs the clinician of the antibiotics that can be used, as well as those drugs that the bacteria is resistant to. This provides a range of responses to determine the most effective treatment, even if it means testing more than one drug.
One person’s story
With thousands of lab tests making their way through the Bacteriology Lab each month, it’s important to remember that each one represents a patient who needs help.
Barrante recalls her own experience with severe illness as a child, and how it inspired her to get into the field of Microbiology.
She remembers the discomfort of getting her blood drawn frequently, and feeling like a “human pin cushion.”
Since being treated, Barrante says she has always wanted to give back, and becoming a medical technologist was the way to do it; performing tests that guide the doctors who treat patients.
“I wanted to help people because I was sick and somebody in a lab somewhere figured out what was wrong with me,” she said.
In photos
Top: A medical technologist inoculates an agar plate with a small amount (approximately 1 microliter) of urine. The plate will incubate in a warm environment to allow bacteria to grow.
Second from top: E. coli bacteria shown in this culture is consistent with what causes most urinary tract infections.
Third: A Haemophilus bacterial infection is visible in the lower plate, which is a media called chocolate agar due to its color.
Second from bottom: Medical technologist Marie Rouse holds a respiratory virus panel, a multiplex PCR test that will run through an automated testing instrument.
Bottom: The Kirby-Bauer disk diffusion test determines what antibiotics are most effective in fighting the bacteria that has grown in culture.