---
# Protocol metadata
id: PROT-0031
name: Preparation of Complete DMEM (High Glucose) Protocol
version: 1.0
description: Protocol for preparing complete DMEM high glucose culture medium with FBS and antibiotics
author: Lab Staff
created: 2025-05-07
last_updated: 2025-05-07
category: media-preparation

# Materials required
materials:
  - name: DMEM high glucose medium
    amount: 500 mL bottle
    storage: 4°C
    notes: Allow to warm to room temperature before use
  - name: Fetal Bovine Serum (FBS)
    amount: 50 mL (10% final)
    storage: -20°C (stock), thaw at 4°C overnight
    notes: Heat-inactivated if required by cell line
  - name: Penicillin-Streptomycin (PS)
    amount: 5 mL (1% final)
    concentration: 100X stock solution
    storage: -20°C
  - name: Sterile 50-mL tube
    type: Polypropylene
    quantity: 1

# Equipment required
equipment:
  - name: Biosafety cabinet
    certification: Class II
  - name: Pipette controller
    type: Electric or manual
  - name: Serological pipettes
    sizes: 5 mL, 10 mL, 25 mL
  - name: Refrigerator
    temperature: 4°C
  - name: Marker or label maker
    use: For labeling media bottles and tubes

# Protocol steps
steps:
  - step: 1
    action: "Prepare workspace"
    details: "Turn on biosafety cabinet, disinfect surfaces, and allow DMEM and supplements to reach room temperature"
  - step: 2
    action: "Prepare plain DMEM aliquot"
    details: "Transfer ~51 mL of DMEM high glucose media into a sterile 50-mL tube and label as 'PLAIN DMEM'"
  - step: 3
    action: "Add FBS to main bottle"
    details: "To the remaining 449 mL of media, add 50 mL Fetal Bovine Serum (FBS) for 10% final concentration"
  - step: 4
    action: "Add antibiotics to main bottle"
    details: "Add 5 mL Penicillin-Streptomycin (PS) to the main bottle for 1% final concentration"
  - step: 5
    action: "Mix gently"
    details: "Invert bottle several times to mix thoroughly without creating bubbles"
  - step: 6
    action: "Label bottle"
    details: "Label the bottle with 'Complete DMEM (High Glucose)', date of preparation, and your initials"
  - step: 7
    action: "Storage"
    details: "Store complete medium and plain DMEM at 4°C for up to 1 month"

# Critical parameters
critical_parameters:
  - parameter: "Sterility"
    details: "Maintain sterile technique throughout to prevent contamination"
  - parameter: "FBS quality"
    details: "Use high-quality, tested FBS batch to ensure consistent cell growth"
  - parameter: "Temperature"
    details: "Cold media can cause cellular stress; allow to warm before use with cells"

# Troubleshooting
troubleshooting:
  - problem: "Medium contamination"
    solution: "Check sterile technique, biosafety cabinet function, and supplement sterility"
  - problem: "Cell growth issues"
    solution: "Test different FBS batches; some cell lines may require additional supplements"
  - problem: "Phenol red color change"
    solution: "Check pH; yellowing indicates acidification which may suggest contamination"

# Safety considerations
safety:
  ppe: "Lab coat and gloves required"
  hazards: "Low biohazard risk; follow institutional guidelines for handling cell culture materials"

# Quality control
quality_control:
  - check: "Visual inspection"
    criteria: "No cloudiness, particles, or contamination"
  - check: "pH indicator (phenol red)"
    criteria: "Should be red-pink color (neutral pH)"
  - check: "Test on standard cell line"
    criteria: "Expected growth rate and morphology"

# References
references:
  - "Cell culture media manufacturer's recommendations"
  - "Laboratory standard operating procedures for media preparation"

# Notes
notes: |
  - This protocol creates both complete DMEM (with FBS and antibiotics) and plain DMEM
  - Complete medium contains 10% FBS and 1% Penicillin-Streptomycin
  - For sensitive cell lines, consider using lower antibiotic concentrations or antibiotic-free media
  - Media shelf life is typically 1 month when stored at 4°C
  - Consider aliquoting complete media to minimize contamination risk from repeated opening
  - Some specialized applications may require additional supplements (e.g., L-glutamine, sodium pyruvate)
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