Gros1 project protocol

Susceptibility to infection with Candida albicans in females of 23 inbred strains of mice (and C5-allele status in 36 strains)   (2011)

Radovanovic I, Mullick A, Gros P


Project protocol — Contents
Workflow and sampling
Reagents, supplies, and solutions

Definitions and calculations

Workflow and sampling


Procedure accomplished
C. albicans
Data Collected
C. albicans strain SC5314 blastospores prepared for infecting mice
incubator, centrifuge
Mice infected via tail vein

200 µL 5 x 104 blastospores

19G needles, 3mL syringes,
Kidneys harvested and processed 48h following infection
dissecting kit, homogenizer, centrifuge
Kidney homogenates cultured and assessed for fungal load
incubator, cell counter
kidney fungal load
Complement component 5 (C5) allele status determined (Hc gene)
C5-allele status


  • Hemocytometer
  • Cell culture incubator heated at 30°C
  • Small rodent dissecting kit
  • Tissue homogenizer
  • Centrifuge
  • Cell/colony counter

Reagents, supplies, solutions

  • YPD medium (1% yeast extract, 2% Bacto Peptone and 2% dextrose)
  • Phosphate-buffered saline (PBS)
  • YPD-agar plates containing 34 mg/mL of chloramphenicol
  • 3 mL syringes
  • 19 gauge needles
  • Cell culture plates
  • Cell culture tubes


I. Infection with Candida albicans and assessment of kidney fungal load
a. C. albicans strain SC5314 is grown overnight in YPD medium at 30°C.
b. Blastospores are harvested by centrifugation, washed twice in PBS, counted using a hemacytometer and resuspended in PBS at the required density.
Female mice are inoculated via the tail vain with 200 µL of a suspension containing 5 x 104 C. albicans blastospores in PBS.
Kidneys are aseptically removed 48h after infection and homogenized in 5 mL of PBS.
e. Kidney homogenate is serially diluted and plated on YPD-agar plates containing 34 mg/mL of chloramphenicol.
f. Plates are incubated at 30°C for 24–48 h and the colony-forming units (CFU) counted and expressed as log10CFU per kidney (maximum sensitivity for this assay is 100 CFU; mice displaying titers below the detection limit are assigned an arbitrary value of 100 CFU).
g. Strains are designated susceptible (=0) or resistant (=1) based on CFU strain means, where susceptible is assigned when log10CFU per kidney > 5.1; resistant is assigned when log10CFU per kidney < 4.2 (Note: designations for susceptible (=0) or resistant (=1) for each strain are assigned to individual animals in the dataset).

II. Evaluating C5 status
Strains are determined to be C5-sufficient or C5-deficient (loss of function): C5 allele status is determined by genotyping genomic tail DNA obtained from JAX® (Note: C5 allele status was determined on one DNA sample for each strain; genotype information is provided for individual mice in the dataset although individual mice in the study were not genotyped). For more information about the C5 deletion, see Wetsel et al.; for information about C5 primers and other procedural details, see Tuite et al.

Submitting investigator's notes: "Despite being C5-sufficient and producing serum C5a during infection (data not shown), SM/J mice display a significantly higher mean fungal load (log10CFU = 5.2 ± 0.4) when compared to the reference strain, C57BL/6J (log10CFU = 4.1 ± 0.5), or other C5-sufficient strains. AKR/J mice are C5-deficient, yet they showed mean log10CFU counts at 3.9 ± 0.4 similar to that seen in the resistant C57BL/6J strain, and clearly distinct from that seen in the C5-deficient group (log10CFU>5.1)."

Data collected by investigator

• fungal load in the kidney

• susceptibility to candidiasis

• complement component 5 (C5) allele status

Definitions and calculations

Candidiasis: mycotic or fungal infections of yeasts (i.e. Candida albicans) origin.

Complement component 5 (C5): is a protein component of the complement system, which when activated results in complement cascade critical for pathogen clearance.

Colony forming units (CFU): is a measure of viable bacterial or fungal numbers.

C. albicans susceptible group = log10CFU > 5.1 (per kidney)

C. albicans resistant group = log10CFU < 4.2 (per kidney)



    Wetsel RA, Fleischer DT, Haviland DL. Deficiency of the murine fifth complement component (C5). A 2-base pair gene deletion in a 5'-exon. J Biol Chem. 1990 Feb 15;265(5):2435-40. PubMed 2303408