Microbiology/LMQA

Topic: Blood Culture

Posted by: Azhar Hamdan TG01 (0503269C)

Equipments & Materials:

• BACTEC™ Fluorescent series blood culture system
• BACTEC™ Plus Aerobic/F Culture Vials Soybean-Casein Digest Broth (Blue Cap)
• BACTEC™ Plus Anaerobic/F Culture Vials Soybean-Casein Digest Broth (Gold Cap)
• Aerobic venting unit
• Class II Biosafety Cabinet
• Calibrated inoculating loops
• Blood Agar Plate (BAP)
• MacConkey Agar (MAC)
• CDC Anaerobic Blood Agar (CDC)
• Sterile 70% ethanol pads (i.e. Alcohol Swab)
• Glass slides
• Gram stain (Crystal violet, Gram’s Iodine[Mordant], Acetone[Decolouriser], Safranin)
• Tubes of pre-prepared 1mL saline
• Tubes of pre-prepared 200µL rabbit plasma

For this posting, I am focusing more on the sub-culturing of blood onto plates. For more information on the BACTEC machine and how it works, refer to BMTjournal (Boon Ching’s Posting on Blood Culture). Linked with permission from Boon Ching.

Basically, the flow would be, BACTEC™ machine THEN sub-culturing of positive vials.

Procedures

Detection:

When the BACTEC machine detects a positive sample, the positive vial (which contains blood and can either be under Aerobic or Anaerobic conditions) is taken out. This is done through the scanning of a barcode at the door of the machine to indicate to the machine that you are removing a vial, followed by the scanning of the vial’s barcode.

The vials have 2 barcodes; one barcode is the original pre-generated BACTEC™ barcode while the other barcode is a pre-generated barcode that is provided by the lab. The laboratory barcodes comes in sets of 2. One of the pair is pasted onto the bottle and the other is pasted onto the request forms.

Following the removal of the vial, the request form is singled out, and qualified staff with access to the Laboratory Information System (LIS) will flag the vial as positive.

BACTEC machine interior

Barcodes located at the door of the machine. These are scanned to tell the machine whether you are adding vials or whether you are removing positive or negative vials

Sub-culturing:

After flagging, 4 labels identical to the vial’s lab barcode numbers are generated. Each of the labels is pasted onto BAP, MAC, glass slide and 1mL saline tube. For an Anaerobic vial, an additional label is generated, with an ‘A’ after the number and this is to be pasted onto a CDC plate. (e.g. DB43210 for the 4 labels, and DB43210A for the Anaerobic plate)

Before anything is done, the cover of the vial and the glass slide is wiped with alcohol swabs. The glass slide is marked with a circle to indicate later on, the area of the drop of blood.

All of the materials, including the vial, are placed in a Biosafety Cabinet (BSC), and the whole process of sub-culturing is done within the BSC. This is to prevent the accidental exposure to aerosols from the blood.

A venting unit is attached to the vial, which would allow the blood to be slowly dripped out for testing. Firstly, 1 drop of blood is dripped each on BAP and MAC. For an Anaerobic vial, an additional drop of blood is dripped onto a CDC plate. Next, an inoculum with 6-fold dilution is obtained by adding 8 drops of blood to the tube of 1mL saline (8drops is roughly 0.2mL). Lastly, 1 drop of blood is dropped onto a glass slide and the drop is spread with an inoculating loop within the pre-indicated circle. Sometime 2 different specimens can be dropped on the same slide to avoid wastage.

The BAP, MAC and CDC (if used) are to be streaked later on after the drop of blood has partially dried. Furthermore, the BAP is streaked with Staphylococcus aureus. BAP and MAC are incubated at 35° in CO₂ for 24 hours whereas the CDC is incubated under anaerobic conditions at 35° for 48 hours before reading. The inoculum (saline with 8 drops of blood) is passed to the investigation lab to be further tested. The blood on the glass slide is allowed to dry on a hotplate within the BSC, before it is gram stained.

Method for streaking
(Also shows the S. aureus streaks done on BAP plates)

BAP that have been incubated at 35° in CO2 for 24 hours

Slides for Gram Stain (Left: Single slide, Right 2 specimens on one slide)

Gram Staining

• Stain with Crystal Violet for 1min
• Flush with water
• Counter-stain with Gram’s Iodine for 1 min
• Flush with water
• Decolourise with Acetone for 2 sec
• Flush with water
• Stain with Safranin for 1 min
• Blot dry

After staining, a presumptive preliminary identification of the organism is done on the basis of the organism being either gram positive or negative or whether it is either a bacilli or cocci. This preliminary identification is crucial for doctors as it allows them to start the patient on the relevant antibiotics.

Direct Tube Coagulase Test

This is done for Gram Positive Cocci (GPC) as a confirmatory test to check whether the GPC is S. aureus. 60 µL of blood (about 3 drops) is added to 200µL rabbit plasma and incubated for 4 hours to overnight.

As S. Aureus is capable of coagulating rabbit plasma, a positive coagulase test would suggest that the GPC is S.Aureus. Further tests are done in the investigation lab, to confirm the identification of S. Aureus.

Thats all for this posting, feel free to ask me questions. Images posted here were taken with permission from laboratory supervisor. He actually encourages us to take photos because he believes that visuals are more important in learning therefore naturally me and the others have been taking pictures non-stop, a fair bit of which is totally unrelated to learning, haha. Anyways, lets all enjoy the rest of our remaining 17 weeks of SIP :) .