McFarland Standards-Based Spectrophotometry Method for Calculating Approximate Multiplicity of Infection for an Obligate Intracellular Bacterium Anaplasma phagocytophilum.

Abstract

Anaplasma phagocytophilum is an obligate intracellular Gram-negative bacterium that causes human granulocytic anaplasmosis. Assessing the number of these bacteria is important for in vitro and in vivo infection studies. Colony count is used to set references for the multiplicity of infections in the case of culturable bacteria. However, the number of bacteria present inside the host cells, in which the bacteria are maintained, can be considered in the case of obligate intracellular bacteria. McFarland standards are a series of turbidity-based standards used to visually assess the approximate number of culturable bacteria. The turbidity of each standard can be related to their respective absorbances or optical densities (ODs). In this study, we describe a simple method to assess the approximate number of A. phagocytophilum based on McFarland standards. The ODs of cell-free crude extracts of A. phagocytophilum were used to assess the approximate number of bacteria while considering that the cell debris also contributes to the ODs. The consistency of this method was also tested using the bacterial cultures grown at different times. In summary, we provide a simple method to estimate the number of obligate intracellular bacteria for use in in vitro infection studies.