Salmonella stained antigen has been widely used in Malaysia for detection of Salmonella infection in poultry. Growth phase of four Salmonella enterica serovar Pullorum (SP 9-25, SP 14/11, SP 690/79 and SP 7107/07) used in the antigen production were investigated based on colony enumeration and turbidity. This study aimed to determine the growth curve and the difference between S. Pullorum isolates based on turbidity measurement and spread plate technique for optimisation towards biomass production of salmonella antigen using bioreactor. Current production of the antigen used conventional methods and the number of bacterial cells is low and with several other drawbacks. The isolates were cultured in nutrient broth, incubated aerobically with constant shake for 48 hours to determine the lag, exponential, stationary and the death phase of the bacteria. Turbidity of the bacterial cells was measured using spectrophotometer and the colony was counted using total plate count every four hours. Based on the colony forming unit per milliliter, SP 690/79 strain showed the fastest growth where this bacteria achieved its mid-exponential growth at 8 hours. This is followed by SP 14/11 where this strain demonstrated the mid-exponential growth at 12 hours. The other two strains (SP 9-25 and SP 7107-07) are the slowest growth where their mid-exponential growth was measured at 14 hours. However, SP 690/79also the fastest strain entering the death phase which demonstrates the difference growth of the S. Pullorum strains. This study demonstrates that each S. Pullorum strains multiplying and dying at different phase though in the same serovar.