Part III—Tracking the Culprit After the microbiologist fi nished Gram staining t
ID: 213136 • Letter: P
Question
Part III—Tracking the Culprit
After the microbiologist fi nished Gram staining the specimens and had examined them, Dr. Maddison
sat down to see if his assessment of the specimen agreed with the microbiologist’s. He peered into
the microscope, straining to see if he could fi nd any clues there to the mystery. As his eyes focused,
he began to see clusters of purple spheres amid some cells that appeared to be a mixture of squamous
epithelial cells and neutrophils. “Gram positive cocci!” he exclaimed. “Now which genera of clinically
signifi cant bacteria are Gram positive cocci that could cause an infection of the skin?” he said to no one
in particular.
“Staphylococcus, of course,” responded Anne Mosel. “And Streptococcus. They’re two of our most
popular isolates from skin specimens.”
Mark found the idea of bacteria being “popular” an odd one, especially bacteria that could cause the
nasty lesions he had seen in the nursery. Moreover, both staph and strep struck him as too tame to be
causing the problem. The fl esh eating bacterium, Streptococcus pyogenes, fell into that group though.
Perhaps it would be an open-and-closed case after all. Mark shuddered as he imagined the reporters who
would storm the clinic once word leaked out to the media that the Black Rock babies were being “eaten
alive.”
“I was just thinking about media,” Mark remarked to Anne. “We can use pretty typical media to isolate
those, right?”
“Yes, we would usually set up these specimens on fi ve percent sheep blood agar, MacConkey agar, and
CNA or PEA agar. We also frequently set up a liquid culture along with the plates.”
“That sounds like a good plan. I’ll check back fi rst thing in the morning to see if there’s any growth on
the plates.” Dr. Maddison thanked Anne and the microbiologist who had helped him and went back upstairs
for a last check on the sick babies before heading home for some much-needed rest.
Upstairs, he nearly ran into Bill Alkin, the hospital administrator who had given him an ultimatum
earlier. “Hello, Bill,” Dr. Maddison said tersely. “What brings you out of the administrative suite for a
second time today?”
“There are some mighty agitated parents in the nursery, Mark. I think you need to convince them that
you have a reasonable plan for taking care of their kids.” Bill turned and began walking back toward his
offi ce. “Remember,” he called over his shoulder to Mark. “Controlling this outbreak is only half of your
problem—controlling the damage is also your concern.”
Mark looked through the window of the nursery in order to size up the situation before entering. He saw
a well-dressed couple in their mid-30s standing beside the Willis twins’ cribs. The woman was quietly
weeping while the man had his arm protectively wrapped around her. Nurse Benoit was standing next to
Baby Girl Levi’s isolette, deep in conversation with a woman with red hair wearing a chenille bathrobe.
The woman was clearly agitated.
Resisting the urge to turn on his heel and fl ee, Mark pushed open the door and entered the nursery. The
couple next to the Willis twins continued their mournful vigil without seeming to notice his presence.
Mark heard Jill say to the woman in the bathrobe, “Here’s Dr. Maddison now. I’m sure he has some
information for you.”
Mark approached the woman with his right hand extended. “I’m Dr. Maddison. You must be Mrs. Levi.
Congratulations on the birth of your daughter.”
“Where’s Dr. Elridge?” said the woman, ignoring his outstretched hand. “She’s taken care of my other
two. No offense, but I want my own doctor taking care of Lilly.”
“Lilly—what a beautiful name,” Mark said, hoping to put Mrs. Levi at ease. “Dr. Elridge is out of the
country for a few weeks. She left me in charge. I’ve already ordered some tests to help us determine the
best treatment for Lilly.”
“Treatment? Just what did she catch from those other babies? Her skin has this terrible rash that’s peeling
off! She looks awful. What kind of a place is this anyway?” The color rising in Mrs. Levi’s face
was beginning to match that of her hair. “If Dr. Elridge can’t take care of her, I’m taking her to Whitley
Memorial Hospital right now.”
Dr. Maddison took a deep breath and responded in a measured tone. “I’m sorry, Mrs. Levi, but I can’t
allow you to take Lilly right now. She’s too sick to be discharged, and Whitley won’t admit her into their
nursery in this condition anyway.”
“You can’t keep my baby here! I’m calling my brother-in-law fi rst thing in the morning. He’s editor of
the biggest newspaper in the county. He’ll make sure people know what kind of a clinic you’re running.”
“Let’s try not to panic, Mrs. Levi. The microbiology lab is zeroing in on the cause of your daughter’s
illness even as we speak.” A bit of an exaggeration, Mark knew, but not an outright lie. “I’ll stop by your
room in the morning and let you know what we’ve found. I’m sure we’ll have this solved by then.”
“You better have it solved by then. I’ll be waiting to hear from you, Dr. Maddison—but I won’t be waiting
for long.” Mrs. Levi turned, pushed open the nursery door, and walked out.
Mark sighed. “Would you please fi ll in the Willis parents?” he said to Nurse Benoit. Jill looked stunned by the
exchange she had just observed. Surprisingly, she simply nodded and walked over to the couple standing by
the cribs and gently began speaking with them. Mark took the opportunity to escape, walking quickly to the
clinic exit and out into the cold night air. Tomorrow would be here sooner than he wanted.
Questions
1. Did your assessment of the Gram stain agree with Dr. Maddison’s? If not, can you explain
why your answer might have been different?
2. Assuming that the organisms are indeed Gram positive cocci, tell the purpose of each of the
media selected by Ms. Mosel to attempt to culture the organism.
3. Which aerobic Gram positive cocci are normal fl ora on the skin?
4. If aerobic Gram positive cocci grow on the plates, what initial tests should be performed to identify
them?
Explanation / Answer
1. Gram staining is an important procedure in microbiology. This differential staining procedure is used to separate most bacteria into two groups based on their cell wall composition.
Gram-positive bacteria has thick layer of peptidoglycan that makes up to about 90% of their cell wall. These bacteria stain purple.
Gram-negative bacteria has thin layer of peptidoglycan that makes up to only 10% of their cell wall and has high lipid content. These bacteria stain pink.
Bacteria usually come in three shapes: spherical, round or oval (cocci), rod shaped (bacillus) and spiral (spirilla).
Dr. Maddison observed clusters of purple spheres in the Gram-stained specimen.
Purple color suggests the presence of Gram-positive bacteria and spherical shape suggests them to be cocci.
Hence, according to me, Dr. Maddison’s assessment of the pathogen to be Gram-positive cocci seems to be true.
2. Different types of media are used in microbiology laboratory.
Blood agar is an enriched bacterial growth medium used to culture those bacteria or microbes that do not grow easily. These bacteria are referred to as “fastidious” as they require a special enriched nutritional environment to grow unlike the routine bacteria. It also helps to differentiate bacteria based on their hemolytic properties. Blood agar is selective for certain pathogens like Streptococcus pyogenes by adding antibiotics or dyes.
MacConkey agar is a selective differential medium commonly used for isolation of Gram-negative bacteria. Since this media possess both selective and differential properties, it can be used to grow group of organisms of interest and helps the investigator to recognize differences in the group based on visible reaction with the media. This media is a powerful diagnostic tool in medical and environmental settings.
Phenyethyl alcohol agar (PEA) agar is a selective medium used to cultivate Gram-positive organisms. Phenyethyl alcohol inhibits the growth of Gram-negative organisms by interfering with DNA synthesis.
Columbia colistin-nalidixic acid (CNA) agar is also a selective medium for Gram-positive organisms. It is especially used to selectively isolate Gram-positive cocci such as staphylococci and streptococci.
Hence, all the media selected by Ms. Mosel is ideal for culturing Gram-positive cocci.
3. Varied environment of the skin results in locally dense or sparse population of bacteria with Gram-positive organisms dominating.
Staphylococcus spp. are normal flora of the skin and mucous membrane. But they can cause disease when introduced into normally sterile sites of the body.
4. Gram staining differentiates Gram-positive and negative bacteria. Gram-positive cocci are seen on Gram stain from a blood culture. The organism grows as beta-hemolytic colony. Further tests that could be performed include:
Catalase test is used to determine whether a microbe produces the enzyme catalase. This enzyme catalyzes the release of oxygen from hydrogen peroxide (H2O2). Streptococcus is catalase negative and Staphylococcus and micrococcus spp. are catalase positive. Catalase test confirm the isolates represent Streptococci.
Pyrrolidonyl Arylamidase (PYR) test is a rapid test which is used for the identification of group A beta-hemolytic Streptococci and Enterococci.
Bacitracin test is used to determine the effect of a small amount of bacitracin on an organism. Streptococcus pyogenes is inhibited by small amount of bacitracin; other beta-hemolytic streptococci usually are not.