Use of Selective and Differential Media

There are many different types of media that are used in a microbiology laboratory. Generally speaking there are three types of media used; selective, differential, and supportive or nutritive.

Selective media are manufactured to support the growth of one type of microorganism while inhibiting the growth of another, in other words, it selectively grows one type of microorganism. It is not uncommon or this media to contain antimicrobials, dyes such as crystal violet, or even alcohol. Some of the more routine selective media types are EMB agar, mannitol salt agar, MAC, and a PEA agar.

Differential media is used to distinguish microorganisms from one another based on growth characteristics that are evident when growth is obtained. There are visible differences between microorganisms when growth is achieved. One fo the more common differential media used is the MacConkey agar. This differentiates between lactose fermenters and non-lactose fermenters.

Supportive media is used to support the growth of a wide range of microorganisms. They are typically non-selective because they want to achieve growth of a wide array of microorganisms. Some more common ones include the sheeps blood agar, as well as the chocolate agar. It has the added X and V factors to support the growth of Haemophilus influenzae.

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Streptococcus Identification and Work Up

Streptococcus is a genus of coccus gram positive bacteria. They typically grow in chains of pairs as cell division occurs along a single axis in this family of bacteria. Most species in the streptococcus genus are oxidase negative, catalase negative, and most are facultative anaerobes. Many streptococcal species are not intact pathogenic and are part of the commensal human microbiota of the skin, mouth, intestine and upper respiratory tract. However, certain streptococcus species are the pathogenic agent for many cases of pink eye, meningitis, bacterial pneumonia, endocarditis, erysipelas, and necrotizing fasciitis.

strep

Species of Streptococcus are classified based on their hemolytic properties. Alpha-hemolytic species cause oxidation of the iron within the red cells, giving a greenish color to appear on the blood agar. The bacteria produces hydrogen peroxide which oxidizes the hemoglobin to biliverdin. This is also referred to as incomplete hemolysis or partial hemolysis. Beta-hemolytic streptococci cause complete degradation of the red cells, appearing as wide clear areas surrounding the bacterial colonies. Gamma-hemolytic species cause no hemolysis.

The beta-hemolytic species are further classified by the Lancefield grouping. This is a serotype classification that describes the specific carbohydrate present on the bacterial cell wall. There are serotypes for Lancefield groups A-V. For times sake, the only ones that will be discussed are Group A, and Group B.

Alpha-Hemolytic Strep

Strep pneumoniae, often referred to as pneumococcus is the leading cause of bacterial pneumonia. It can also be the etiological agent for otitis media, sinusitis, meningitis and peritonitis. The viridian’s group of alpha-hemolytic streptococci are a large group of commensal organisms. They possess no Lancefield antigens (carbohydrates) and can or can not be hemolytic.

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Beta-Hemolytic Strep

Streptolysin which is an exotoxin is the enzyme produced by the strep species that causes complete hemolysis of the red cells in the media. There are two types of streptolysin; Streptolysin O (SLO) and Streptolysin S (SLS). Streptolysin O is an oxygen-sensitive (labile) cytotoxin which is secreted by most of the Group A Streptococcus (GAS) which interacts with cholesterol in the membrane of the red cells. Streptolysin S is an oxygen-stable cytotoxin also produced by GAS species that affects the innate immune system of the host. It works in preventing the host immune system from clearing the infection.

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Group A Strep

Group A strep, otherwise known as S. pyogenes is the causative agent for strep infections, invasive and non-invasive. The most common infection is pharyngitis, otherwise known as strep throat, impetigo, and scarlet fever. All these infections are non-invasive. Invasive GAS infections include necrotizing fasciitis, pneumonia, and bacteremia (bacteria present in the blood). Complications can arise from GAS infections. Rheumatic fever is a disease that affects the joints, kidneys, and the heart valves. It is the consequence of an untreated strep A infection. Antibodies created by the immune system cross-react with proteins in the body which causes an immune-mediated attack on the hosts own cells. Essentially an acquired auto-immune disease. GAS is also implicated in pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS). Autoimmune antibodies affect the basal ganglia causing rapid onset of psychiatric, motor, sleep and other neurological symptoms, primarily in the pediatric population.

GAS is diagnosed by either an rapid strep test or by culture.

Group B Strep

GBS, otherwise known as S. agalactiae, causes pneumonia and more importantly meningitis in neonates and the elderly. The American Congress of Obstetricians and Gynecologists now recommends that all women between 35 and 37 weeks gestation to be tested for group B strep. Those who test positive should be given prophylactic antibiotics during labor. The bacteria can cause premature rupture of the membranes during pregnancy and can colonize and cross the placenta to infect the fetus.

Laboratory diagnosis and Workup

After initial culture and preliminary identification of beta-hemolytic streptococcus is suspected there are further identification tests to make an accurate diagnosis. The first is by Lancefield antigen determination. Commercially available Lancefield antisera is used for the differentiation of beta-hemolytic species. These usually come as small kits and are directed to Lancefield groups A, B, C, F, and G. Antigen detection is demonstrated by agglutination by specific antibodies that are provided.

The PYR test is a rapid colormetric method most often used to distinguish S. pyogenes from other beta-hemolytic species. The PYR tests for the presence of the enzyme pyrrolidonyl aminopeptidase. This enzyme hydrolyzes L-pyrrolidonyl-b-naphthylamide (PYR) to B-naphthylamide, which produces a red color when a specific reagent is added. The test can be performed on paper strips that contain dried chromogenic substrates for the pyrrolidonyl aminopeptidase. S. pyogenes is PYR positive and in the case of an unknown organism displaying S. pyogenes morphology plus being PYR positive, it is acceptable to presumptively identify as S. pyogenes.

Bacitracin susceptibility is another test that can be used to differentiate S. pyogenes from other beta-hemolytic strep species. S. pyogenes has an increased susceptibility to bacitracin. A pure culture must be obtained and streaked on a sheep blood agar plate. A small disk containing 0.04 U of bacitracin is placed on the plate and incubated overnight at 35 degrees celsius in 5% CO2. A zone of inhibition surrounding the disk indicates susceptibility.

With the introduction and advancement of nucleic acid detection and serological methods in the laboratory it is now getting easier and faster to detect strep species without relying on culturing and further confirmation testing. It should be important to note that this does not replace the use of a culture or any of the further testing mentioned above. Serological testing relies on antibodies to anti-streptolysin O and anti-DNase B. The antibody levels against streptolysin O rise within one week of infection and peak around 3-6 weeks. DNase B is a nuclease among many that S. pyogenes uses to escape neutrophil extracellular nets. DNase B is specific for S. pyogenes. The antibody levels to DNase B rise post two weeks infection and reach maximum titers at around 6-8 weeks.

The Optochin test is used to differentiate alpha-hemolytic streptococci. This is either S. pneumoniae or strep viridians. S. pneumoniae species are sensitive to the chemical ethylhydrocupreine hydrochloride, otherwise known as optochin. Optochin disks, sometimes called P disks can be obtained from a commercial vendor. A pure culture is obtain and incubated to allow growth overnight. When growth has occurred, a subculture is plated and a P disk is placed on the agar and incubated overnight at 35 degrees celsius with 5% CO2. The zone of inhibition is then measured and a zone greater than 14 mm indicates sensitivity and allows for the presumptive diagnosis of pneumococci.

The bile solubility test is usually used as confirmatory test for S. pneumoniae that distinguishes it from other alpha-hemolytic species. Sodium deoxycholate (Bile) will lyse the cell wall of pneumococcal species.

-Caleb

Urinalysis

A urinalysis is exactly what the name entails. An analysis of a patients urine. A urinalysis is a fairly common test that may be ordered as part of an annual physical or part of diagnostic testing. It can be used as an evaluation of UTIs, Diabetes Mellitus, kidney disease, kidney stones, proteinuria, rhabdomyolysis, liver disease, or if a patient presents with particular symptoms such as abdominal pain, flank pain, painful urination, blood upon urination, and fever. Pregnancy testing is also part of a routine urinalysis if ordered.

The first step in an urinalysis is collection of the specimen from the patient. An optimal sample is an early morning sample, as it is the most concentrated produced during the day. There are no fasting requirements or medication schedule dosage changes unless otherwise directed by the patients ordering physician. 30-60 mL of urine is collected in a clean urine specimen cup through a clean catch method that should be explained to the patient upon request of sample.

There are different variants of urinalysis. One is the macroscopic observation of the urine. This is the direct visual observation which includes noting its quantity, clarity, and color. Urine is normally yellow and clear without any cloudiness. Abnormalities can mean different things and further analysis is needed.

Cloudy: Infection

Dark Yellow: Dehydration

Brown: Liver disease (caused by an accumulation of bilirubin)

Red: Blood (Indicates UTI, stones, tumors, renal trauma)

Orange/Tea Colored: Rhabdomyelitis (Breakdown of muscle)

Foamy: May suggest excess protein

It is important to note that certain medications taken for UTIs can change the color of the urine, Phenazopyridine in particular.

Macroscopic -Urinalysis

Dipstick chemical analysis is performed on a narrow plastic strip which has individual tests denoted with different colored squares. The entire strip is dipped into the urine sample and color changes of the squares are noted either by the technologist or by inserting it into an instrument to read it. Each square takes a specific amount of time to react so its important to allow the reaction to come to fruition before resulting. The color change of particular squares are compared to a reference guide and can point out abnormalities.

In no particular order the squares on the dipstick indicate;

Specific gravity (concentration of the urine)

pH

Protein concentration

Glucose concentration

Presence of Ketones

Hemoglobin

Leukocyte esterase (Suggestive of WBC in urine)

Nitrite (Suggestive of bacteria in urine)

Bilirubin

Urobilinogen

Multistix-Urinalysis-Color-Key

Dipsticks are convenient and are easy to interpret and cost-effective. Its important to keep in mind that dipsticks are qualitative and not quantitative in that they only suggest that there is an abnormality, they don’t quantify that abnormality. Further analysis is required for such results.

Microscopic analysis can detect cells, cellular debris, bacteria, crystals, and certain casts to confirm the dipstick results and further quantify analysis. Once the samples are received they must be centrifuged and discarding the supernatant. Epithelial cells may suggest inflammation or damage to the gallbladder and casts and cellular debris suggest inflammation of the kidneys and upper urinary tract. On very rare occasions tumor cells can be seen which are diagnostic for certain renal carcinomas and other urinary tract cancers.

If red cells are noted it could indicate either an infection, trauma, or stones. They can also indicate glomerulonephritis which is inflammation of the kidneys. Sometimes small amounts of red cells will be seen in healthy individuals.

Urine is considered a sterile body fluid therefore there should be no white blood cells or bacteria. Any amount of WBC or bacteria within the urine is considered abnormal and is suggestive of an UTI, cystitis, or pyelonephritis.

Identifying crystals if any is important and lend diagnostic information as to what is pathologically going on within the body.

Uric acid crystals can vary in size and shape, but usually resemble a rhomboid shape. These crystals are common in individuals with urate nephrolithiasis or acute urate nephropathy.

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Cystine crystals are usually colorless hexagonally shaped and look similar to benzene rings (bringing it back to organic chemistry days). These occur in patients with cystinuria which is a genetic defect in renal cystine transport and in acidic urine (pH <6.0).

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Struvite crystals are often described as having a coffin-lid appearance. These crystals are typically magnesium ammonium phosphate and are seen in alkaline urine (pH >7.0). Seen in patients with UTIs caused by urea-splitting bacteria (Proteus mirabilis) or in patients with infected calculi (struvite stones).

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Calcium oxalate crystals are typically found in acidic urine and can take on multiple shapes. Some may look like colorless ovoids, biconcave discs, or even rods. Usually seen in patients with high dietary oxalate ingestion, patients with nephrolithiasis or those in ethylene glycol toxicity with renal failure.

Triple-phosphate-crystal

These are some of the more common crystals that will be seen. Urinalysis isn’t flashy and isn’t always fun as it is someones urine, but it is an important part of routine testing to better deliver care to the patient.

-Caleb

 

 

Blood Draw Tube Colors and Order

The tube order may not seem like a big deal and may seem unnecessary to some, but it is very important to pay attention too. It also matters as to what type of needle is being used for the draw. If a butterfly needle is being used it is important to have a spit tube because with a butterfly there is air within the hose that connects the needle to the vacutainer. Its important to get this air out before filling any tubes used for patient testing. If a standard needle is being used, you typically don’t need the spit tube, but its good practice. The order still remains the same for each.

Light Blue: The typical tube for routine coagulation studies. The additive is sodium citrate (3.2% or 3.8%). Citrate is a anticoagulant which binds to calcium within the blood so the blood can’t clot. Calcium plays an important role in primary and secondary homeostasis. See my post on DIC for that information, in short it is used in the coagulation cascade. An important aspect of coagulation studies is that the light blue tube must, must be filled completely. There is a ratio of sodium citrate to whole blood and that must remain constant. The tube must be rejected if it is not filled completely.

Green or Mint Tubes: These tubes are used for chemistry studies. Often referred to as PST or plasma separator tubes. The additive in these tubes are sodium heparin, lithium heparin or ammonia heparin. The heparin, being an anticoagulant activates antithrombin, which blocks the coagulation cascade and produces a whole blood with plasma sample instead of a clotted blood and serum sample. When these tubes are centrifuged, the gel barrier moves upwards creating a barrier that separates the plasma from the red cells allowing the plasma to be aspirated directly for testing.

Gray Tube: The gray tube tops are typically used for glucose testing, ethanol levels or lactate level testing. The additive is potassium oxalate and sodium fluoride. Potassium oxalate is an anticoagulant which prevents clotting and the sodium fluoride is an anti-glycolytic which prevent the cells from using the glucose in the sample.

Lavender/Pink Tube: The lavender tube is typically used for hematological testing or for Hemoglobin A1C testing. The pink tube is used primarily for blood bank testing such as type and screen and cross-matching. The additives in the lavender and pink tubes are EDTA K2 or EDTA K3. The EDTA binds to calcium which blocks the coagulation cascade in the same way that citrate in the light blue tube does. Red cells, leukocytes, and platelets are in EDTA anticoagulated blood for 24 hours. Blood smears should be done within 3 hours of receiving the sample.

SST/Mustard Tube: Serum separator and clot activator that will separate the blood from the serum upon centrifugation. This tube is usually used to test for aldosterone, B12, ferritin and folate levels.

There are not all the different tubes that are used, but these are the most common tubes that I listed and the ones that a laboratory professional will most likely come across. Its important to understand the additive in each one to make sure that they are appropriate for the testing that needs to be done on the patient sample itself within the tube.

phb_Order of Blood Draw with labels72dpi

-Caleb

37-year-old South American Male Case Study Mini-Series

The purpose of this mini-series is to get in the mind of a treating physician when a patient such as this presents to the clinic or the ED in this case. The first part of this series is the introduction of the case with case history and initial lab testing. Please don’t hesitate to leave comments on what you think the diagnosis is and what other confirmatory tests need to be done if any as well as what treatment should consist of. This is mean’t to stimulate a discussion and there are no wrong answers. I am in no way a physician or at that level or have that education. I am a student with a passion for molecular diagnostics and creating these cases is a good way for me to practice real life scenarios through careful and diligent research as well as help others who think the same way. This case is no way real and all lab values are made up to the best of my knowledge. If anything is incorrect please do not hesitate to email me or  leave a comment.

CASE:

A 37-year-old South American male presented to his annual physical with his primary care physician with general fatigue, decreased appetite and weight loss over the past three weeks. The patient mentioned to his physician that he has had multiple nosebleeds throughout the last few weeks, an occurrence of multiple a week. The patients past medical history is unremarkable. No family history of bleeding tendencies. He is not taking any prescription medication and denies use of recreational drugs and only social use of alcohol. His physician ordered a CBC and a prothrombin time/activated partial thromboplastin time (PT/aPTT). Results are in table 1.

Two days later the patient presented to the emergency room with fever and heavy fatigue, he explained to the attending that it has been hard to do anything the last few days, and has been bed-ridden. Physical exam revealed bilateral bruising on the upper arms and forearms with purpura and petechiae. The attending physician ordered a full coagulation panel, platelet function tests (Ristocetin cofactor assay), bleeding time test for vWD, and full CBC with peripheral blood smear analysis. Results are summarized in table 2.

Later that evening the patient developed a high fever, and back/flank pain and was moved to the ICU. Blood cultures, CRP and a procalcitonin was ordered, results are in table 3.

Positive cultures for Staphylococcus aureus were found after 48-96 hours and the patient was started on a course of vancomycin and monitored closely.

Patient results indicated he was pancytopenic with a hemoglobin of 9.7 g/dL (Ref. 13.5-18.0 g/dL) and RBC count of 3.7×10^3/uL (Ref. 4.20-6.00×10^6 uL) with severe thrombocytopenia at 37×10^3/uL (Ref. 150-450×10^3/uL).

Initial coagulation results revealed significantly elevated PT and aPTT. The bleeding time test along with the results from the RCO indicate platelet dysfunction or acquired inhibition of platelets by accelerated destruction. Platelet aggregation studies were normal. RCO studies indicate factor VIII inhibition or consumption.

The peripheral blood smear confirmed leukopenia and thrombocytopenia and revealed abnormal promyelocytes with abundant azurophilic granulation and multiple auer rods in bundles. RBC morphology showed schistocytes and fragmented cells.

The attending followed up by ordering a complete fibrinogen, D-dimer and a plasminogen panel. Results are in table 4.

The significantly elevated D-dimer, elevation in t-PA and u-PA in combination with the significant decrease in fibrinogen, and plasminogen levels indicates primary hyperfibrinolysis.

The attending sent a blood sample to the Blood Bank laboratory and asked for units of packed red cells, platelets, and fresh frozen plasma (FFP) to be transfused. With the additional blood components, the patient was able to regain control over the thrombocytopenia, hemoglobin, fibrinogen and coagulation factor levels.

A bone marrow aspirate was ordered including cytology, cytochemistry, immunophenotyping, FISH (Fluorescence in situ hybridization), cytogenetics (chromosomal analysis and FISH) and RT-PCR for PML/RARA quantification of transcripts. The attending started the patient on all-trans retinoic acid (ATRA) as induction therapy.

FISH revealed the PML-RARA fusion gene present which was later quantified and confirmed by RT-PCR. PCR sequencing revealed a bcr-3 PML-breakpoint. Chromosomal analysis of the bone marrow identified a t(15;17) classic translocation. Cytochemistry revealed intensely positive reacting cells to myeloperoxidase and Sudan black B. Immunophenotyping results are in table 5.

Table 1:

RBC: 4.10×10^6/uL             4.20-6.00×10^6/uL          

HGB: 12.9 g/dL                    13.5-18.0 g/dL        

HCT: 38.7%                          40-54%

MCV: 88 fL                            80-100 fL

MCH: 33.2 pg                        26-34 pg

MCHC: 32.3 g/dL                  32-36 g/dL

RDW: 13.5%                         11.5-14.5%

RETIC: 0.8%                          0.5-2.5%

NRBC: 0/100 WBC               0

WBC: 6.3×10^3/uL              3.6-10.6×10^3/uL

NEUT: 3.6×10^3/uL             1.7-7.5×10^3/uL

LYMPH: 1.9×10^3/uL          1.0-3.2×10^3/uL

MONO: 0.7×10^3/uL           0.1-1.3×10^3/uL

EO: 0.1×10^3/uL                  0.0-0.3×10^3/uL

BASO: 0                                 0.0-0.2×10^3/uL

PLT: 111×10^3/uL              150-450×10^3/uL

MPV: 7.3 fL                           7.0-12.0 fL

 

PT: 21 seconds                    11-14 seconds

aPTT: 37 seconds               25-35 seconds

Table 2:

RBC: 3.7×10^3/uL              4.20-6.00×10^3/uL

HGB: 9.7 g/dL                     13.5-18.0 g/dL                    

HCT: 28.9%                          40-54%

MCV: 71 fL                            80-100 fL

MCH: 31.8 pg                       26-34 pg

MCHC: 33.1 g/dL                 32-36 g/dL

RDW: 15.1%                        11.5-14.5%

RETIC: 2.3%                         0.5-2.5%

NRBC: 0/100 WBC               0

WBC: 2.5×10^3/uL             3.6-10.6×10^3/uL

NEUT: 1.3×10^3/uL           1.7-7.5×10^3/uL    

LYMPH: 0.7×10^3/uL         1.0-3.2×10^3/uL

MONO: 0.3×10^3/uL          0.1-1.3×10^3/uL

EO: 0.1×10^3/uL                 0.0-0.2×10^3/uL

BASO: 0.1×10^3/uL            0.0-0.3×10^3/uL

PLT: 37×10^3/uL               150-450×10^3/uL

MPV: 19.3 fL                       7.0-12.0 fL

 

Myeloblasts: 7%                  0%

Promyelocytes: 54%         0%

Myelocytes: 3%                    0%

Metamyelocytes: 5%           0%

Bands: 0%                             0%

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PT: 33 seconds                   11-14 seconds

aPTT: 63 seconds              25-35 seconds

BT: 13 minutes                   1-9 minutes

RCO: 30%                              50-150%

Platelet aggregation studies: Normal

Table 3:

Blood Cultures: POS Staph aureus          NEG

Procalcitonin: 0.25 ng/mL                        <0.15 ng/mL

CRP: 23 mg/L                                                0-10 mg/L

Table 4:

Fibrinogen: 67 mg/dL

Plasminogen: Reduced

a2-Antiplasmin: Reduced

t-PA: Elevated

u-PA: Elevated

D-Dimer: >19,000 ng/mL    

Table 5:

CD2                NEG

CD4                NEG

CD13              POS

CD14              NEG

CD16              NEG

CD19              NEG

CD33              POS

CD34              NEG

CD45              POS

CD56              NEG

CD64              POS

CD117            POS

HLA-DR         NEG

 

-Caleb