Unknown Bacterial Identification *Notes* Micro. Lab - Crider

Your Gram stain is complete and correct. Which of the following statements would apply to the image you see?

You can observe gram-positive cocci on this field.

After incubation with a novobiocin disc, you observe the plate results. Your plate is shown on the right, with a control plate for novobiocin sensitivity on the left. What can you conclude?

Your plate shows no zone of inhibition and the unknown bacterium can be classified as resistant to novobiocin.

a metabolically active and growing cell

vegetative cell

Gram-negative Bacilli----positive oxidase---negative glucose fermentation----positive nitrate reduction =

Pseudomonas aeruginosa

Wound and blood stream infections, complications of burns, complications of cystic fibrosis, skin rashes, corneal (eye) ulcers, meningitis, endocarditis, urinary tract infections

Pseudomonas aeruginosa

One initial suspect bacteria you considered was the common sepsis (bloodstream infection) organism Staphylococcus aureus. Why can we rule this organism out as the cause in the patient?

The Gram stain result shows the unknown organism is gram-negative. As S. aureus is a known gram-positive organism, it can be ruled out.

One initial suspect bacteria you considered was common endocarditis bacterium Enterococcus faecalis. Why can we rule this organism down as the cause of the patient?

The Gram-stain result shows the unknown organism as gram-positive cocci in clusters, suggestive of staphylococci. E. faecalis is a streptococcus species and would appear as a gram-positive chain, and hence can be ruled down.

gram negative bacilli----negative oxidase-----positive lactose fermentation---negative methyl red----negative motility =

Klebsiella pneumoniae

Urinary tract infections, opportunistic infections related to fecal contact

Alcaligenes faecalis

Over the past 2 days, Jenny, an active healthy 3 yr-old who had successfully potty-trained a year earlier, began to have "accidents" along with appearing in pain and straining when going to the bathroom. Her mom noted that Jenny's urine had a faint pink tinge. A trip to the pediatrician quickly confirmed that Jenny had a urinary tract infection (UTI). A urine specimen was collected and sent to the lab for identification. The lab performed a quantitative culture and pure culture. The next morning, the lab called the pediatrician, indicating that Jenny's urine quantitative culture grew >100,000 CFU/mL of bacteria. The lab team also isolated the organism into pure culture and was provided to you on a nutrient agar plate and in a liquid broth culture tube for further investigation. Based on the information; what organism(s) is suspected?

Alcaligenes faecalis Enterococcus aerogenes Escherichia coli Proteus vulgaris

includes observing bacterial morphology, staining properties, and biochemical testing results. These are done visually noting the appearance, color, etc. of a test

Phenotypic characteristic

bacterial cell shape that is cylindrical or rod-like (longer than it is wide)

Bacillus

urinary tract infection

Enterobactor aerogenes

Wound infections, urinary tract infections

Proteus vulgaris

urinary tract infection

Staphylococcus saprophyticus

bacterial cell shape that is spherical or rounded in appearance

coccus

You correctly selected the catalase test as your next step. Which of the following is TRUE regarding the catalase test?

-The catalase results will help you to move down the dichotomous key. -According to the dichotomous key, catalase activity is present in some bacteria and absent in others.

Which is TRUE regarding the oxidase test?

-The oxidase results will help you to move down the dichotomous key. -According to the dichotomous key, oxidase activity varies in certain organisms

Which of the following is/are TRUE regarding labeling this plate?

-The plate must be labeled with as much information as known at this time because we are working with unknown organisms. Accurate and appropriate labels are important in cases of accidental exposures. -The label should be placed on the bottom (agar side) of the plate to keep lid condensation from falling onto the agar surface, avoiding contamination, and to ensure sample accuracy in case lids are switched

Internationally recognized reference book and collection of datasets for bacterial classification and identification

Bergey's Manual of Determinative Bacteriology

refers to the analysis of the bacterial DNA sequence for unique orders that are found in only one organism. This method is highly sensitive but subject to contamination and errors

Genetic characteristic (genetic characterization)

What results are from this gram stain?

Gram-negative Baccilus

visual flowchart that can be used to map the series of testing that help identify an organism. Each step has two outcomes, positive or negative, leading to the next step. The top would consider a broad spectrum of organisms. The possibilities narrow at each level/step until arriving at a single possibility at the bottom.

Dichotomous key

urinary tract infections and surgical wound infections

Enterococcus faecalis

acute diarrhea, urinary tract infections, pneumonia, wound and bloodstream infections, meningitis, infections of the newborn

Escherichia coli

gram-negative bacilli----negative oxidase---positive lactose fermentation---positive methyl red---- positive indole =

Escherichia coli

For most of the morning, Tracey was experiencing low pelvic pain and burning sensation each time she urinated. She recognized the symptoms of a bladder infection and made an appointment with an urgent care clinic for later that day. A urinalysis showed an abundance of white blood cells, suggesting a urinary tract infection (UTI). A specimen of clean-catch urine was sent to the microbiology lab where a sterile calibrated loop was used to deliver and streak out 1 microliter of urine on an agar plate, which yielded colonies too numerous to count. An inoculation and overnight incubation of the urine into sterile broth yielded heavy growth. Based on the information in the case, what organism(s) could or should be suspected to cause this scenerio?

Escherichia coli Staphylococcus saprophyticus Streptococcus agalactiae Staphylococcus aureus

Why is it important to observe and record results of your phenol red broth (PRB) immediately after 24 hours of incubation?

Extended incubation can lead to reversion and inconclusive test results

Three-year-old Jason woke up at 2 AM, crying and pulling on his right ear. These symptoms were similar to those he had last month and again three months earlier. In both cases, he was diagnosed with otitis media (ear infection) and was treated with antibiotics. The next day, the pediatrician explained that she suspected an antibiotic-resistant strain of bacteria and performed a tympanocentesis (sample of infected fluid from behind the eardrum) procedure for bacterial culture to determine exactly what microorganism was causing the infection. An additional antimicrobial susceptibility test to determine the most appropriate antibiotic for treatment was ordered also. The ear fluid specimen was sent to you In the clinical microbiology lab for ID. Upon receipt, the specimen was cultured on agar media and grown in a pure culture for your determination. Based on the information in the case, what organism(s) could or should be suspected to cause this scenario?

Haemophilus influenzae Streptococcus pneumoniae Streptococcus agalactiae

The identification of an unknown organism by antibody binding assays. This method is rapid and specific, but subject to false positives and negatives as well as relying on the immune activity of the patient or sample

Immunologic characteristic

Pneumonia, wound and bloodstream infections, meningitis, urinary tract infections, meningitis, urinary tract infections

Klebsiella Pneumoniae

After addition of nitrate reagents A and B, your tube remains colorless. Is your testing for nitrate complete?

No, testing is incomplete because the unknown bacterium may reduce nitrate in the tube to other nitrogenous forms besides nitrite or not reduce the nitrate in the tube at all.

When swimming in the creek, Anna slipped and a rock cut a three-inch gash in her leg. The next day, the area around the cut was swollen and red and the pain was increasing. When she began to run a fever later in the afternoon and the wound began to throb, a friend took her to the emergency room. A specimen of pus was collected from the wound for the culture. An IV was started, delivering a broad-spectrum antibiotic. Blood work showed high activity and numbers of white blood cells. Quick work by the laboratory team isolated the organism into pure culture and was provided to you on a nutrient agar slant and in a liquid broth culture tube for further investigation Patient condition is becoming critical. Unknown bacterium identification needed STAT. Based on the information in the case, what organism(s) could or should be suspected to cause this scenario?

Pseudomonas aeruginosa Escherichia coli Staphylococcus aureus

acute diarrhea, osteomyelitis (bone infections)

Salmonella typhimurium

Gram negative bacilli--- negative oxidase---negative lactose fermentation ---- positive H2S production----negative urease =

Salmonella typhimurrium

Pneumonia, otitis media (ear infections), complications of burns, wounds, and blood stream infections, meningitis

Serratia marcescens

Following hip replacement surgery, 90 year-old James was anticipating his transfer to rehabilitation center, when he suddenly spiked a fever of 38.5 degrees C (normal temp is 37) He started experiencing chest pain and difficulty breathing. The attending physician noticed some crackles and abnormal lung sounds by stethoscope exam. A chest X-ray showed fluid accumulating in his lungs, a clear sign of lower respiratory tract infection and pneumonia. A sputum specimen was collected in a sterile container and promptly delivered to the lab. The sample was marked STAT for unknown organism id before Jame's lung function was compromised and he would need to be put on a ventilator. The lab isolated the organism into pure culture and it was provided to you on a nutrient agar plate and in a liquid broth culture tube for further investigation. What are the suspects?

Serratia marcescens Escherichia coli Klebsiella pneumoniae Streptococcus pneumoniae

acute diarrhea, dysentery (bloody diarrhea)

Shigella sonnei

Emma and her friend packed a picnic lunch, including cold-cut chicken sandwiches, egg salad, and a Thermos with fresh cold, unpasteurized milk from her father's farm. Two days later, both Emma and her friend began to experience nausea and vomiting, which progressed to include watery diarrhea. With no signs of improvement 48 hours later. Emma visited her physician. After a brief physical exam revealing signs of dehydration. Emma's doctor recommended oral rehydration with Pedialyte and collected stool specimen for culture. The sample was sent for unknown id of the cause of the gastrointestinal illnesses. The lab team isolated that organism into pure culture and it was provided to you on nutrient agar and in a liquid broth culture for further investigation. What organism(s) are suspects?

Shigella sonnei Escherichia coli Salmonella typhimurium

The severe storm had damaged the sewage treatment plant, which was now flooded and leaking raw sewage into the nearby creek. This creek emptied into a large lake that serves as the water supply for a metropolitan area and surrounding counties. Local agencies tasked with monitoring water quality noted greatly increased numbers of bacteria. Individuals that drank water from the contaminated reservoir developed nausea and vomiting, along with diarrhea, which could appear bloody (dysentery). Samplers were taken from affected patients and sent for unknown organism identification of the cause of these gastrointestinal illnesses. The lab team isolated the organism into pure culture and it was provided to you on a nutrient agar plate and in a liquid broth culture tube for further investigation. What organism(s) is suspected?

Shigella sonnei Salmonella typhimurium Escherichia coli

Gram-negative bacilli-----negative oxidase---negative lactose fermentation----negative H2S production----negative citrate =

Sigella sonnei

round shaped bacterial cells arranged in irregular clusters

Staphylococcus

Gram-positive Cocci---positive Catalase----positive MSA---positive coagulase =

Staphylococcus aureus

skin infections, abscesses, wound infections, pneumonia, urinary tract infections, endocarditis and septicemia

Staphylococcus aureus

Miriam was in her 38th week of pregnancy and was surprised when after just a few mild contractions, her water broke. This was followed by a long labor, after which she delivered a healthy baby boy. About 12 hours later, the newborn became feverish with a temperature of 38.8 degrees C. He also had an elevated heart rate and rapid shallow respirations. Suspecting possible bloodstream (sepsis) infection, the medical team ordered blood cultures, which were sent to you in the microbiology lab. Twenty-four hours later, there was evidence of growth in the culture tubes. Colonies were further isolated on an agar plate for additional investigation and identification. Based on the information in the case, what organism(s) could or should be suspected to cause this scenario?

Staphylococcus aureus Streptococcus agalactiae Escherichia coli

Gram-positive cocci ---- positive catalase ---- negative MSA ---- Sensitive Novobiocin =

Staphylococcus epidermidis

Skin and joint infections, endocarditis, urinary tract infections, and septicemia

Staphylococcus epidermidis

For the past 10 days, Jackie felt exhausted and lacked her usual stamina. Several times, she ran a fever, which abated when she took acetaminophen. Just walking up the stairs left her short of breath. Since the symptoms seemed to be worsening, she visited her physician. The nurse practitioner noted that her heart murmur, present since childhood, was noticeably louder and much more obvious. She had also developed a petechial rash (purplish streaks) under her finger nails. She had a fever of 38.2 degrees C at the time of the visit. When asked about her activities over the last month, she mentioned that she had a minor outpatient orthopedic surgery to check her previously surgically replaced knee. Suspecting possible bloodstream infection and endocarditis, the medical team ordered blood cultures, which were sent to you in the microbiology lab. Within 24-36 hours later, there was evidence of growth in the culture tubes. Colonies were further isolated on an agar plate for additional investigation and identification. Based on the information in the case, what organism(s) could or should be suspected to cause this scenario?

Staphylococcus epidermidis Staphylococcus aureus Enterococcus faecalis

Gram-positive cocci---- positive catalase--- negative MSA --- Resistant Novobiocin =

Staphylococcus saprophyticus

Round shaped bacterial cells arranged in chains

Streptococcus

Gram-positive cocci----negative catalase test----Beta hemolysis on blood agar----Resistant Bacitracin----positive CAMP =

Streptococcus agalactiae (Group B Strep)

Urinary tract infections and infections of the new born

Streptococcus agalactiae (Group B strep)

Pneumonia, otitis media (ear infections), conjuctivitis, and meningitis

Streptococcus pneumoniae

gram-positive cocci----negative catalase---alpha hemolysis blood agar plate----sensitive optochin =

Streptococcus pueumoniae

Strep throat, impetigo, scarlet fever, and necrotizing fascitis

Streptococcus pyogenes

Annie was outdoors and received multiple mosquito bites. She scratched a lot, even to the point that some had begun to bleed. Several of Annie's mosquito bites were not healed well, and instead had now become swollen and painful, suggesting an infection. When the infection didn't respond to several applications of triple antibiotic ointment, Annie's mother took her to the pediatrics clinic. A sterile swab was used to collect a specimen of pus from the infected site, which was sent to the lab for isolation and stock culture creation. After overnight incubation, the specimen showed significant bacterial growth. Based on the information in the case, what organism(s) could or should be suspected to cause this scenario?

Streptococcus pyogenes Staphylococcus epidermis Staphylococcus aureus

We now have a Gram stain result. Why can we NOT make a conclusive diagnosis yet as to the cause in our patient?

The Gram stain result shows the unknown organism is gram-negative. All our possible suspects (E. coli, E. aerogenes, A. faecalis, and P. vulgaris) are gram-negative, and hence cannot be ruled out without further differentiation

One initial suspect bacteria you considered was the common sepsis (bloodstream infection) organism. Streptococcus pyogenes. Based on the Gram stain result, why can we rule this organism down as the cause in the patient?

The Gram-stain results suggest the unknown organism is gram-positive cocci in clusters. The appearance for S. pyogenes would be expected to be gram-positive cocci in chains, and hence could be ruled down as less likely.

The mannitol salt agar (MSA) test selects bacterial growth in high salt conditions and also differentiates mannitol fermentation by detecting acidic products. Here are the results of your MSA test. The plate on the right is your sample compared to a control reaction plate on the left. What can you conclude?

The MSA test shows the unknown bacterium grew and is negative for mannitol fermentation

The mannitol salt agar (MSA) test selects for bacterial growth under high-salt conditions and also differentiates mannitol fermentation by detecting acidic products. Here are the results of your MSA test. The plate on the right is your sample compared to the control reaction plate on the left what can you conclude?

The MSA test shows the unknown bacterium grew and is negative for mannitol fermentation.

The mannitol salt agar (MSA) test selects for bacterial growth under high-salt conditions and also differentiates mannitol fermentation by detecting acidic products. Here are the results of your MSA test. The plate on the left is your sample compared to a control reaction plate on the right. What can you conclude?

The MSA test shows the unknown bacterium grew and is positive for mannitol fermentation. (The organism grew and caused medium to change color to yellow)

MacConkey agar helps detect lactose fermentation into acidic products and is used to classify bacteria based on this result for an unknown organism. Here are the results of your MacConkey agar growth. What can you conclude?

The MacConkey agar test shows the unknown bacterium is negative for lactose fermentation

MacConkey agar helps detect lactose fermentation into acidic products and is used to classify bacteria based on this result for an unknown organism. Here are the results of your MacConkey agar growth. What can you conclude?

The MacConkey agar test shows the unknown bacterium is positive for lactose fermentation

The H2S test in TSI agar slants detects the formation of hydrogen sulfide by bacteria and is used to classify bacteria. based on this result for an unknown organism. Here are the results of your H2S test. What can you conclude?

The TSI slant colors show the unknown bacterium is positive for H2S production

The H2S test in TSI agar slants detects the formation of hydrogen sulfide by bacteria and is used to classify bacteria based on this result for an unknown organism. Here are the results of your H2S test. What can you conclude?

The TSI slant shows no black of any kind and hence the unknown bacterium is negative for H2S production

The blood agar hemolysis test grows bacteria with fastidious conditions and also differentiates between bacterial hemolysis of RBCs by visualization. Here are the results of your blood agar test. What can you conclude?

The blood agar hemolysis test shows the unknown bacterium grew and is alpha-hemolytic.

The blood agar hemolysis test grows bacteria with fastidious conditions and also differentiates between bacterial hemolysis of RBCs by visualization. Here are the results of your blood agar hemolysis test. What can you conclude?

The blood agar hemolysis test shows the unknown bacterium grew and is beta-hemolytic

The catalase test is used to classify bacteria based on the presence or absence of catalase enzyme in an unknown organism. Here are the results of your catalase test. What can you conclude?

The catalase test on the unknown bacteria is negative

The catalase test is used to classify bacteria based on the presence or absence of catalase enzyme in an unknown organism. Here are the results of your catalase test. What can you conclude?

The catalase test on the unknown bacterium is positive. (bubbles = positive)

The citrate test on Simmons agar slants detects the metabolism of citrate by bacteria and can be used to classify bacteria based on this result for an unknown organism. The results of your citrate test are shown in the left tube. A control reaction citrate tube is on the right. What can you conclude?

The citrate test is negative for this unknown organism

After addition of the nitrate reagents A and B, as well as zinc addition, your tube remains colorless. What can you conclude?

The colorless result after zinc addition shows the unknown bacterium reduced nitrate to nitrogenous forms other than nitrite and can be classified as a "nitrate reducer"

The oxidase test is used to classify bacteria based on the presence or absence of cytochrome oxidase enzyme in an unknown organism. Here are the results of your oxidase test. What can you conclude?

The oxidase test on the unknown bacterium is positive

We now have a gram stain. Why can we NOT make a conclusive diagnosis yet as to the cause of our patient?

The gram stain result shows the unknown organism gram-negative. All our possible suspects (E. coli, S. marcescens, an K. pneumoniae) are gram negative and hence cannot be ruled out without further differentiation

We now have a Gram-stain result. Why can we NOT make a conclusive diagnosis yet as the cause in our patient?

The gram stain result shows the unknown organism is gram negative. all our possible suspects (E. coli, S. typhimurium, and S. sonnei) are gram-negative, and hence cannot be ruled out without further differentiation

One initial suspect bacteria you considered was the common UTI-causing bacterium Escherichia coli. Why can we rule this organism down as the cause in the patient?

The gram stain results shows the unknown organisms is gram-positive. E. coli would appear gram-negative and hence can be ruled out.

One initial suspect bacteria you considered was the common otitis media bacterium Haemophilus influenzae. Why can we rule this organism down as the cause in this patient?

The gram-stain result shows the unknown organism as gram-positive cocci chains, suggestive of streptococci. H. influenzae is a gram-negative bacterium, and hence can be ruled down.

The indole test detects the ability of the bacteria to convert tryptophan into indole and is used to classify bacteria based on this result for an unknown organism. Here are the results compared to control reaction on the right. What can you conclude?

The indole test shows the unknown bacterium is positive for tryptophan conversion to indole

The MR-VP test detects glucose fermentation into acidic products and is used to classify bacteria based on this result for an unknown organism. Here are the results of your MR-VP test. The broth on the right is your sample compared to a positive control reaction on the left. WHat can you conclude?

The methyl red test shows the unknown bacterium is negative for glucose fermentation

The MR-VP test detects glucose fermentation into acidic products and is used to classify bacteria based on this result for an unknown organism. Here are the results of your MR-VP test. The broth on the right is your sample, compared to a positive control reaction on the left. What can you conclude?

The methyl red test shows the unknown bacterium is positive for glucose fermentation

The motility test detects the movement of bacteria visually in a semi-solid medium, and is used to classify bacteria based on this result for an unknown organism. The results of your motility test are shown in the right rube. A control motility test is on the left. What can you conclude?

The motility test is negative for this unknown organism

The oxidase test is used to classify bacteria based on the presence or absence of cytochrome oxidase enzyme in an unknown organism. Here are the results of your oxidase test. What can you conclude?

The oxidase test on the unknown bacterium is negative

The Phenol Red broth (PRB) test detects glucose fermentation in acidic products and is used to classify bacteria based on this result for an unknown organism. Here are the results of your PRB test. The broth on the left is your sample, compared to a control reaction on the right. Durham tubes can be disregarded, as no gas production is noted. What can you conclude?

The phenol red test shows the unknown bacterium is negative for glucose fermentation

The urease test slant detects the bacterial conversion of urea to ammonia and can be used to classify bacteria based on this result for an unknown organism. The results of your urease slant test are shown in the left tube. A control reaction urease slant is on the right. What can you conclude?

The urease test is negative for this unknown organism

Cholera (acute, copious secretory diarrhea)

Vibrio cholerae

Your gram stain is complete. Which of these statements would apply?

You can observe gram-negative bacilli on this field

Your gram-stain stain is complete. Which would apply to the image?

You can observe gram-positive cocci

Your gram stain is complete and correct. Which of the following would apply to the image you see?

You can observe gram-positive cocci on this field

Your gram stain is complete and correct. Which of the following would apply to the image you see?

You can observe gram-positive cocci on this field (the purple color indicates a positive test)

After incubation for a CAMP test you observe the plate results. Your plate is shown at the left, with a control plate on the right. What can you conclude?

Your plate results show the unknown bacterium can be classified as CAMP possitive

After incubation with a novobiocin disc, you observe the plate results. Your plate is shown on the right, with a control plate for novobiocin sensitivity on the left. What can you conclude?

Your plate shows a zone of inhibition and the unknown bacterium can be classified as sensitive to novobiocin

After incubation with an optochin disc, you observe the plate results. Your plate is shown at the left with a control plate on the right. What can you conclude?

Your plate shows a zone of inhibition and the unknown bacterium can be classified as sensitive to optochin.

After incubation with bacitracin disc, you observe the plate results. Your plate is shown at the left, with a control plate on the right. What can you conclude?

Your plate shows no zone of inhibition, and the unknown bacterium can be classified as resistant to bacitracin

After addition of bacteria to your coagulase tube, you tilt the tube to observe for any clotting of the rabbit plasma reagent, due to the presence of coagulase enzyme. Your tube is shown on the left with a negative control tube for coagulase on the right. What can you conclude?

Your tube shows the coagulation of rabbit plasma and the unknown bacterium can be classified as positive for coagulase enzyme production.

tests used in the identification of bacterial species because of the inherent differences in physiologic and metabolic activities of bacterial species these reactions can help provide presumptive identification of isolate based on the presence or absence of a trait (catalase, oxidase, sugar fermentation...)

biochemical testing

visible mass of bacteria growing together on an agar plate or slant

colony

From the gram-stain what is the result:

gram positive

Now that a pure culture has been isolated for you, what would be a good starting procedure to begin classifying your unknown organism?

gram-staining