1. Obtaining a Sample of Infected Tissue
For a diagnosis of staph infection, a sample of an infected lesion (nasal secretion, pus, blood, cerebro-spinal fluid, urine, cell aspirate from lungs or bone) has to be obtained. Symptoms of staph infections are often caused by toxins, released from only few staphylococci, so a culture of obviously infected lesion may be negative! From this reason, samples from more than one lesion has to be provided, if possible.
2. Gram Staining
A clinical sample is placed on a slide and rinsed with:
- Crystal Violet
- Iodine Solution (the Mordant)
- Decolorizer (ethanol)
- Safranin (the Counterstain)
After this procedure, S.aureus and other staphylococci should be blue, purple or violet under the light microscope, what is noted as Gram-positive (Gram +), (Picture 1). Bacteria that are colored light pink are noted as Gram negative (Gram -), (1). Gram staining is performed only, when mixed infection with Gram + and Gram – bacteria is expected.
Picture 1. Gram positive S.aureus,
light microscope, Magn. ~ 100x
3. Staphylococcus aureus Culture
When staph is expected, a part of a medical specimen is spread over the trypticase soy agar (TSA) with 5% sheep blood (BAP = blood agar plate), (2). On such agar, S.aureus forms golden yellow colonies (cream white to orange), (3), while S.epidermidis forms white colonies (Picture 2). Culture usually needs 16-18 hours to grow (up to 48 hours for blood culture) at 35°C (4). S.aureus is usually hemolytic on sheep blood, and S.epidermidis is not. In mixed specimens, containing Gram + and – bacteria, a Columbia agar based CNA plate (colisitin/naladixic acid) to suppress Gram negative growth may be used.
Picture 2. Staph culture:
golden S.aureus and white S.epidermidis colony.
4. Enzyme Tests
a) Catalase test is performed by adding 3% hydrogen peroxide to a colony on agar. Staphylococci contain catalase, and break down peroxide, produces O2 and bubble, so they are catalase positive, what distinguish them from streptococci. Catalase test is done only when a culture is not typical.
b) Coagulase test. S.aureus (and S.intermedius, which is not pathogenic for human) causes coagulation (fibrin clot) of a rabbit plasma, so it is coagulase positive, which distinguish it from all other staphylococci. In latex agglutination test (slide test), latex particles covered with fibrinogen and IgG are used. IgG antibodies bind with coagulase on S.aureus, what results in latex particles clumping in about 20 seconds (5). This slide test may be negative in a small percent; in this case a tube test, which detects both free and bound coagulase, has to be performed (5). About 97% of human S. aureus isolates possess both forms of coagulase (6).
c) Hemolysis. S.aureus causes hemolysis on a blood agar, but S.epidermidis does not. Some S.aureus strains are not hemolytic though (7).
5. Rapid Diagnosis of Staphylococcus aureus with PCR
Rapid detection of S.aureus is possible by tests using Quantitative Polymerase Chain Reaction (qPCR), also called Real-time PCR.
Principle of PCR
In clinical samples, S.aureus is present in quantities not sufficient to be exactly determined by any testing method, so bacteria have to be multiplied by culturing, what usually takes 1-4 days. With a PCR, a few million of copies of S.aureus DNA can be obtained directly from a clinical sample (no culture needed) within a few hours.
A sample of staph DNA, polymerase – an enzyme needed for DNA duplication, a sequence of nucleotids (primer), needed for the start of duplication, and a large amount of free nucleotids are needed for PCR. Polymerase is obtained from a bacterium Thermus aquaticus (Taq polymerase). All components are added into a testing tube, which is put into a thermal cycler. After some PCR cycles, agarose gel electrophoresis and southern blotting technique are used to determine if obtained DNA is S.aureus DNA (8).
6. Staphylococcus aureus Detection Kits
Kits for detection of various species of staphylococci or their toxins from human tissues, food, cow’s milk, water, and other environmental sources, are commercially available.
Examples of the kits, based on agglutination, for detection of MRSA in clinical samples (after culturing) are: Dry Spot Staphytect Plus®test, Pastorex Staph Plus®test, the Slidex Staph-Kit®,Slidex Staph Plus®test, the StaphaurexPlus®test and the Staphylase Test®. Specificity and sensitivity of these kits was evaluated by three German health institutes in y. 2005 (9).
Xpert™ MRSA/SA-Blood Cultures (BC) and Xpert™ MRSA/SA-Skin and Soft Tissue Infection (SSTI) tests are able to simultaneously detect MRSA and S.aureus directly from blood cultures and samples of infected soft tissue, using PCR. Both tests give results in approximately 50 minutes (10).
Foodmay be tested for staph enterotoxins by ELISA. Presence of S.aureus bacteria in the food may be confirmed by TAQMAN (PCR), or Slidex Staph Kit (latex agglutination) (11).
Milk can be tested for cow’s mastitis by Bulk Tank Culture (BTC), California Mastitis Test (CMT), and specifically for staph mastitis by Prostaph© milk antibody test (12). The multiplex real-time PCR assay for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk was developed (13). Test takes 24 hours to be completed.
Water, in order to be tested for S.aureus, is filtered and then the filtering membrane is put on modified mannitol salt agar for culturing (14).
S.aureus from an infected lesion has to be cultured on a solid medium and then appropriate suspension prepared from few grown colonies. Suspension with bacteria is swabbed over a Mueller-Hinton agar (picture 3), (15). Discs impregnated with various antibiotics are placed onto agar and incubated for 24 hours at 35 °C. Zones of inhibition of bacterial growth, measured around discs, should be compared with those in the National Committee for Clinical Laboratory Standards (NCCLS) Document M100 (M2). Results obtained may then be reported as resistant, intermediate or susceptible.
Picture 3. S.aureus antibiotic sensitivity test.
Areas of inhibited bacterial growth are seen around antibiotic discs.
Microbiology Report Form for S. aureus
Below are three examples of Microbiology Report, filled by microbiologist after laboratory testing, and sent back to the patient’s doctor.
a) Report for a drainage from a skin carbuncle, infected by S. aureus:
Source (anatomical location): Drainage from the neck abscess
Gram staining: Many gram positive cocci. Heavy growth. Many WBCs (presence of white blood cells indicates inflammation).
Organism: Staphylococcus aureus
*MIC (Minimal Inhibitory Concentration) = the lowest concentration of antimicrobial agent that inhibits the growth of the microbe. MIC is determined by adding increasing amounts of antibiotic (titration method) to medical sample.
**SENS = sensitivity; S = sensitive, R = resistant.
*** Bacteria in above sample were susceptible to oxacillin, so this is methicillin susceptible SA (“normal” SA). To determine methicillin resistance, oxacillin is used instead of methicillin, because methicillin is no longer commercially available in the United States, and because oxacillin maintains its activity during storage better than methicillin (16).
b) Report for blood, infected by MRSA:
Gram staining: Many WBCs. Few gram positive cocci. Light growth.
Organism: Staphylococcus aureus
* High dose of oxacillin was needed to inhibit bacteria, so this is considered as a methicillin resistant staph – MRSA.
c) Report of nasal swab, COLONIZED by S. aureus:
Source: Nasal mucosa
Gram staining: Gram positive cocci. Light growth. No WBCs*
Organism: Staphylococcus aureus
- What is Staphylococcus aureus?
- What is Staphylococcus epidermidis?
- Staph Epidemiology
- MRSA Superbug Types
- Microorganisms – Types, Pictures
- Gram staining (dcccd.edu)
- Microbiology lab expert (prep4usmle.com, member ‘bactitech’)
- S. aureus is not always yellow (microbiology.mtsinai.on.ca)
- Staph culture growth time (cinetwork.com)
- Stapyloslide test (bd.com)
- Staph virulence factors, superantigen toxins (utmb.edu)
- Non-hemolytic strains of S. aureus (cdc.gov)
- PCR (bionewsonline.com)
- SA/MRSA detection kits (sgmjournals.org)
- SA/MRSA simultaneous rapid test (rapidmicrobiology.com)
- Taqman, kitsfor detection of S. aureus in food (foodhaccp.com)
- Milk tests for staph mastitis (uwex.edu)
- Diagnosis of cow mastitis (fass.org)
- Detection of S. aureus in water (hpa-standardmethods.org.uk)
- Mueller-Hinton agar (bd.com)
- Why is oxacillin used instead of methicillin to detect MRSA? (cdc.gov)